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Circulating EBV-DNA in the monitoring of EBV infection in pediatric liver transplant recipients
Circulating EBV-DNA in the Monitoring of EBV Infection in Pediatric Liver Transplant Recipients M. Spada, M. Guizzetti, W. Petz, M. Colledan, A. Segalin, A. Lucianetti, A. Bertani, G. Peloni, A. Sonzogni, D. Alberti, S. Riva, M. Melzi, and B. Gridelli
E
PSTEIN-BARR VIRUS (EBV) infection can induce uncontrolled lymphocyte B proliferation in immunosuppressed transplant recipients. The risk of developing EBV-related posttransplant lymphoproliferative disease (PTLD) is particularly high in children who contract primary EBV infection after transplantation.1 The incidence of PTLD in the pediatric population following liver transplantation (LTx) can exceed 10%.2 It has been reported that the monitoring of EBV-specific DNA sequences in blood circulation of transplant recipients by polymerase chain reaction (PCR) can help to identify patients at risk of developing PTLD before the onset of clinical signs.3,4 The purpose of this study was to asses the use of quantitative detection of EBV-specific sequences in peripheral blood of pediatric liver transplant recipients for the early identification and for the presymptomatic treatment of EBV infection by immunosuppression reduction or withdrawal.
PATIENTS AND METHODS All pediatric LTx recipients transplanted after October 1997 with known pretransplant EBV serology were included in this prospective study. EBV serology was evaluated before LTx by EBV nuclear antibody (EBNA) levels, determined using the ELISA technique. Transplantation techniques have been described in detail elsewhere.5 After LTx, patients were monitored by EBV DNA quantitation in peripheral blood mononuclear cells (PBMC), performed 1 and 3 months after LTx, and every 3 months thereafter, and for the development of EBV-associated symptoms. The quantitative PCR methods has been described elsewhere.6 Briefly, serial amounts of two recombinant DNA molecules, corresponding to two regions of the EBV EBNA-1 gene, were amplified in the presence of PBMC DNA from transplanted patients. Previous studies carried out on asymptomatic and EBV-symptomatic solid organ recipients7 demonstrated that levels of EBV DNA equal or higher than 500 genomes/105 PBMC are related to a significantly higher risk of EBV-related disease development. Asymptomatic EBV infection was treated by reduction of immunosuppression, with subsequent EBV DNA quantitation performed every 15 days. If after immunosuppression reduction EBV DNA remained high or further increased or EBV-associated symptoms developed, immunosuppression was withdrawn. In patients developing EBVassociated symptoms, in between the scheduled assays, EBV DNA quantitation was performed and EBV localization in tissues was investigated by histology, immunohistochemistry, and/or in situ hybridization methods. EBV-related PTLD diagnosis was based on © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
the histologic criteria of Frizzera et al.8 In these patients, immunosuppression was reduced as described for asymptomatic patients. Postoperative management was according to the established protocol for pediatric LTx. Immunosuppression consisted of cyclosporin (CyA)-based or tacrolimus-based dual therapy, with steroid weaning 6 months after transplantation. CyA target plasma level was 250 to 350 ng/mL during the first 2 weeks after LTx, 200 to 300 ng/mL from week 3 to week 12, 150 to 200 ng/mL from month 4 to month 12, and 50 to 150 ng/mL thereafter. Tacrolimus target whole blood level was 10 to 20 ng/mL in the first 2 weeks, 10 to 15 ng/mL from week 3 to week 4, 5 to 15 ng/mL from month 2 to month 3, and 5 to 10 ng/mL thereafter. Clinical or biochemical suspicion of acute rejection was always confirmed by liver biopsy.
RESULTS
Between October 1997 and July 2000, 95 children underwent 105 LTx. The most frequent indication to LTx was extrahepatic biliary atresia, diagnosed in 58 patients (61%). In nine patients pretransplant EBV serology was not available. These patients and nine others who died in the early postoperative period (within 30 days) were not included in the study. Median age at transplantation of the 77 studied children was 1.8 years (range, 0.2 to 17). Median follow up was 15.5 months (range, 1 to 33). Baseline immunosuppression was with CyA and steroids in 48 patients (62%) and with tacrolimus and steroids in 29 patients (38%). In the CyA group, 14 (29%) patients developed steroid-resistant rejection and were switched to Tacrolimus therapy. Thirtynine patients (51%) were EBNA negative before transplantation, and 38 patients (49%) were EBNA positive. Median age in the two groups was significantly different: 1 year versus 4.5 years (P ⬍ .01). After transplantation, 55 patients never showed EBV DNA levels higher than 500 genomes/105 PBMC. Median age in this group was 4 years (range, 0.2 to 17). Twentythree (42%) were EBNA negative before transplantation, and 32 (48%) were EBNA positive. None of these patients developed symptomatic EBV infection or PTLD during the From the Liver Transplantation Center (M.S., M.G., W.P., M.C., A.S., A.L., A.B., G.P., D.A., S.R., M.M., B.G.) and the Department of Pathology (A.So.), Ospedali Riuniti di Bergamo, Bergamo, Italy Address reprint requests to Marco Spada, MD, PhD, Chirurgia 3, Ospedali Riuniti, Largo Barozzi 1, 24128 Bergamo, Italy. 0041-1345/01/$–see front matter PII S0041-1345(00)02828-1 1835
Transplantation Proceedings, 33, 1835–1837 (2001)
1836
SPADA, GUIZZETTI, PETZ ET AL
Table 1. Incidence of Asymptomatic and Symptomatic EBV Infection in the Patients Clustered by Type of Immunosuppression EBV DNA
⬍500 genomes/10 PBMC ⱖ500 genomes/105 PBMC Asymptomatic Symptomatic 5
Tacrolimus (n ⫽ 29)
Cyclosporin (n ⫽ 27)
Switch (n ⫽ 21)
24 (83%) 5 (17%) 3 (60%) 2 (40%)
17 (63%) 10 (27%) 4 (40%) 6 (60%)
14 (67%) 7 (33%) 5 (71%) 2 (29%)
study period. After a median time from LTx of 7 months, 12 patients (16%) showed levels of EBV DNA equal or higher than 500 genomes/105 PBMC, ranging from 500 to 3000 genomes/105 PBMC, in the absence of EBV-associated symptoms. Median age in this group of patients was 0.8 year (range, 0.4 to 2.5), significantly lower compared to the previous group of patients (P ⬍ .01). Eight were EBNA negative (67%), four were EBNA positive (33%). In all these patients, immunosuppression was reduced achieving stable EBV DNA levels normalization. None of these children experienced EBV-associated symptoms or PTLD. Ten children (13%) developed symptoms of EBV infection in between the scheduled EBV DNA assays at a median time after LTx of 6 months. EBV DNA levels assessed at the time of symptoms development were always higher that 500 genomes/105 PBMC, ranging from 1000 to 10000 genomes/105 PBMC. Median age of this children was 0.8 year (range, 0.5 to 3). Eight (80%) were EBNA negative and two were EBNA positive (20%). Symptoms were fever in five patients, rash in one, lymphoadenopathy in three, upper airway obstruction in four, and gastrointestinal symptoms in five. In two cases EBV was detected in the lymph nodes and tonsils, with a histologic picture of mononucleosis-like syndrome. In all the symptomatic children immunosuppression was reduced and in four cases was stopped later on because of the persistence of high EBV DNA levels. In all but one child, EBV-associated symptoms resolved after immunosuppression modulation, with normalization of EBV DNA levels. One patient, a 3-year-old female who was EBNA-negative before LTx, developed a polymorphic, polyclonal PTLD localized in the liver and tonsils. She was treated by immunosuppression withdrawal, achieving PTLD resolution. Immunosuppression discontinuation caused the development of acute rejection, requiring steroid boluses and reestablishment of immunosuppression, without PTLD recurrence. During rejection treatment the patient contracted chicken pox infection that forced us to reduce the immunosuppression therapy with consequent development of chronic rejection. Of the 19 patients treated by immunosuppression reduction, 3 (16%) experienced at least one episode of acute rejection, requiring steroid boluses treatment and reestablishment of adequate immunosuppressive treatment. Two of these patients developed steroid-resistant rejection and were switched to tacrolimus. In all the other children, immunosuppression was adjusted to the normal range after the normalization of EBV DNA levels. In 4 children immunosuppression was stopped. All experienced acute rejection; one was switched to tacrolimus and in all the
immunosuppression was restarted after a median time of 12 days (range, 7 to 20). One patient developed chronic rejection. The incidence of asymptomatic and symptomatic EBV infection in the study patients stratified by the type of immunosuppression did not revealed significant differences (Table 1). DISCUSSION
EBV infection is common after pediatric LTx and is associated with the risk of PTLD. Despite the identification of risk factors that predispose patients to develop EBVrelated diseases, a recent review emphasized the limitation of our knowledge of its pathogenesis, variable criteria for establishing the diagnosis, and lack of studies addressing the prevention and treatment of EBV-related diseases.9 Early diagnosis and decreased immunosuppression are important steps in the management of EBV infection and PTLD. Circulating EBV genome level determination has been previously reported to be useful in identifying organ and stem cell transplant recipients at increased risk of PTLD development.3,4 We used an EBV DNA quantitation assay for the early diagnosis of EBV infection and for the presymptomatic treatment of the infection by immunosuppression modulation, using a cut off value of 500 genomes/ 105 PBMC. No patients with EBV DNA levels below 500 genomes/105 PBMC developed EBV infection or PTLD. Those who showed levels of EBV DNA equal to or higher than 500 genomes/105 PBMC were treated by immunosuppression reduction and did not developed symptomatic EBV infection or PTLD. Modulation of immunosuppression was effective in preventing the development of symptomatic infection and caused only in few cases acute rejection episodes (16%), successfully treated by steroid boluses and/or conversion to tacrolimus immunosuppression. Chronic rejection developed in only one child and was not directly related to the treatment of EBV infection. In 13% of patients, symptomatic EBV infection developed in between the scheduled EBV DNA assay. Small children, EBNA negative before LTx, largely composed this group of patients. These data confirm those of other investigators who observed that young transplant patients undergoing primary EBV infection are at higher risk of developing EBV-related diseases,4 and suggest that EBV DNA levels should be assessed more frequently than every 3 months. PTLD incidence in our series was 1%, corresponding to the lower reported ranges.1 A higher risk of PTLD development has been reported in patients receiving tacrolimus immunosuppression.2 Our results did not show differences
MONITORING EBV INFECTION
in the incidence of symptomatic EBV infection clustering the patients according to the immunosuppression therapy. Even if the follow-up time of the study is limited, we can conclude that the use of EBV DNA quantitation in the peripheral blood of pediatric liver recipient seemed to have a positive impact in the early identification of EBV infection, allowing to reduce immunosuppression before the onset of symptomatic EBV infection and PTLD. REFERENCES 1. Newell KA, Alonso EM, Whitington PF, et al: Transplantation 62:370, 1996 2. Younes BS, McDiarmid SV, Martin MG, et al: Transplantation 70:94, 2000
1837 3. Riddler SA, Breinig MC, McKnight JLC: Blood 84:972, 1994 4. Krieger NR, Martized OM, Krams SM, et al: Liver Transpl 6:62, 2000 5. Spada M, Gridelli B, Colledan M, et al: Liver Transpl 6:415, 2000 6. Gerna G, Furione M, Baldanti F, et al: J Clin Microbiol 32:2709, 1994 7. Baldanti F, Grossi P, Furione M, et al: J Clin Microbiol 38:613, 2000 8. Frizzera WP, Hanto DW, Gajl-Peczalska KJ, et al: Cancer Res 41:4262, 1981 9. Paya C, Fung JJ, Nalesnik M, et al: Transplantation 10:1517, 1999
E
PSTEIN-BARR VIRUS (EBV) infection can induce uncontrolled lymphocyte B proliferation in immunosuppressed transplant recipients. The risk of developing EBV-related posttransplant lymphoproliferative disease (PTLD) is particularly high in children who contract primary EBV infection after transplantation.1 The incidence of PTLD in the pediatric population following liver transplantation (LTx) can exceed 10%.2 It has been reported that the monitoring of EBV-specific DNA sequences in blood circulation of transplant recipients by polymerase chain reaction (PCR) can help to identify patients at risk of developing PTLD before the onset of clinical signs.3,4 The purpose of this study was to asses the use of quantitative detection of EBV-specific sequences in peripheral blood of pediatric liver transplant recipients for the early identification and for the presymptomatic treatment of EBV infection by immunosuppression reduction or withdrawal.
PATIENTS AND METHODS All pediatric LTx recipients transplanted after October 1997 with known pretransplant EBV serology were included in this prospective study. EBV serology was evaluated before LTx by EBV nuclear antibody (EBNA) levels, determined using the ELISA technique. Transplantation techniques have been described in detail elsewhere.5 After LTx, patients were monitored by EBV DNA quantitation in peripheral blood mononuclear cells (PBMC), performed 1 and 3 months after LTx, and every 3 months thereafter, and for the development of EBV-associated symptoms. The quantitative PCR methods has been described elsewhere.6 Briefly, serial amounts of two recombinant DNA molecules, corresponding to two regions of the EBV EBNA-1 gene, were amplified in the presence of PBMC DNA from transplanted patients. Previous studies carried out on asymptomatic and EBV-symptomatic solid organ recipients7 demonstrated that levels of EBV DNA equal or higher than 500 genomes/105 PBMC are related to a significantly higher risk of EBV-related disease development. Asymptomatic EBV infection was treated by reduction of immunosuppression, with subsequent EBV DNA quantitation performed every 15 days. If after immunosuppression reduction EBV DNA remained high or further increased or EBV-associated symptoms developed, immunosuppression was withdrawn. In patients developing EBVassociated symptoms, in between the scheduled assays, EBV DNA quantitation was performed and EBV localization in tissues was investigated by histology, immunohistochemistry, and/or in situ hybridization methods. EBV-related PTLD diagnosis was based on © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
the histologic criteria of Frizzera et al.8 In these patients, immunosuppression was reduced as described for asymptomatic patients. Postoperative management was according to the established protocol for pediatric LTx. Immunosuppression consisted of cyclosporin (CyA)-based or tacrolimus-based dual therapy, with steroid weaning 6 months after transplantation. CyA target plasma level was 250 to 350 ng/mL during the first 2 weeks after LTx, 200 to 300 ng/mL from week 3 to week 12, 150 to 200 ng/mL from month 4 to month 12, and 50 to 150 ng/mL thereafter. Tacrolimus target whole blood level was 10 to 20 ng/mL in the first 2 weeks, 10 to 15 ng/mL from week 3 to week 4, 5 to 15 ng/mL from month 2 to month 3, and 5 to 10 ng/mL thereafter. Clinical or biochemical suspicion of acute rejection was always confirmed by liver biopsy.
RESULTS
Between October 1997 and July 2000, 95 children underwent 105 LTx. The most frequent indication to LTx was extrahepatic biliary atresia, diagnosed in 58 patients (61%). In nine patients pretransplant EBV serology was not available. These patients and nine others who died in the early postoperative period (within 30 days) were not included in the study. Median age at transplantation of the 77 studied children was 1.8 years (range, 0.2 to 17). Median follow up was 15.5 months (range, 1 to 33). Baseline immunosuppression was with CyA and steroids in 48 patients (62%) and with tacrolimus and steroids in 29 patients (38%). In the CyA group, 14 (29%) patients developed steroid-resistant rejection and were switched to Tacrolimus therapy. Thirtynine patients (51%) were EBNA negative before transplantation, and 38 patients (49%) were EBNA positive. Median age in the two groups was significantly different: 1 year versus 4.5 years (P ⬍ .01). After transplantation, 55 patients never showed EBV DNA levels higher than 500 genomes/105 PBMC. Median age in this group was 4 years (range, 0.2 to 17). Twentythree (42%) were EBNA negative before transplantation, and 32 (48%) were EBNA positive. None of these patients developed symptomatic EBV infection or PTLD during the From the Liver Transplantation Center (M.S., M.G., W.P., M.C., A.S., A.L., A.B., G.P., D.A., S.R., M.M., B.G.) and the Department of Pathology (A.So.), Ospedali Riuniti di Bergamo, Bergamo, Italy Address reprint requests to Marco Spada, MD, PhD, Chirurgia 3, Ospedali Riuniti, Largo Barozzi 1, 24128 Bergamo, Italy. 0041-1345/01/$–see front matter PII S0041-1345(00)02828-1 1835
Transplantation Proceedings, 33, 1835–1837 (2001)
1836
SPADA, GUIZZETTI, PETZ ET AL
Table 1. Incidence of Asymptomatic and Symptomatic EBV Infection in the Patients Clustered by Type of Immunosuppression EBV DNA
⬍500 genomes/10 PBMC ⱖ500 genomes/105 PBMC Asymptomatic Symptomatic 5
Tacrolimus (n ⫽ 29)
Cyclosporin (n ⫽ 27)
Switch (n ⫽ 21)
24 (83%) 5 (17%) 3 (60%) 2 (40%)
17 (63%) 10 (27%) 4 (40%) 6 (60%)
14 (67%) 7 (33%) 5 (71%) 2 (29%)
study period. After a median time from LTx of 7 months, 12 patients (16%) showed levels of EBV DNA equal or higher than 500 genomes/105 PBMC, ranging from 500 to 3000 genomes/105 PBMC, in the absence of EBV-associated symptoms. Median age in this group of patients was 0.8 year (range, 0.4 to 2.5), significantly lower compared to the previous group of patients (P ⬍ .01). Eight were EBNA negative (67%), four were EBNA positive (33%). In all these patients, immunosuppression was reduced achieving stable EBV DNA levels normalization. None of these children experienced EBV-associated symptoms or PTLD. Ten children (13%) developed symptoms of EBV infection in between the scheduled EBV DNA assays at a median time after LTx of 6 months. EBV DNA levels assessed at the time of symptoms development were always higher that 500 genomes/105 PBMC, ranging from 1000 to 10000 genomes/105 PBMC. Median age of this children was 0.8 year (range, 0.5 to 3). Eight (80%) were EBNA negative and two were EBNA positive (20%). Symptoms were fever in five patients, rash in one, lymphoadenopathy in three, upper airway obstruction in four, and gastrointestinal symptoms in five. In two cases EBV was detected in the lymph nodes and tonsils, with a histologic picture of mononucleosis-like syndrome. In all the symptomatic children immunosuppression was reduced and in four cases was stopped later on because of the persistence of high EBV DNA levels. In all but one child, EBV-associated symptoms resolved after immunosuppression modulation, with normalization of EBV DNA levels. One patient, a 3-year-old female who was EBNA-negative before LTx, developed a polymorphic, polyclonal PTLD localized in the liver and tonsils. She was treated by immunosuppression withdrawal, achieving PTLD resolution. Immunosuppression discontinuation caused the development of acute rejection, requiring steroid boluses and reestablishment of immunosuppression, without PTLD recurrence. During rejection treatment the patient contracted chicken pox infection that forced us to reduce the immunosuppression therapy with consequent development of chronic rejection. Of the 19 patients treated by immunosuppression reduction, 3 (16%) experienced at least one episode of acute rejection, requiring steroid boluses treatment and reestablishment of adequate immunosuppressive treatment. Two of these patients developed steroid-resistant rejection and were switched to tacrolimus. In all the other children, immunosuppression was adjusted to the normal range after the normalization of EBV DNA levels. In 4 children immunosuppression was stopped. All experienced acute rejection; one was switched to tacrolimus and in all the
immunosuppression was restarted after a median time of 12 days (range, 7 to 20). One patient developed chronic rejection. The incidence of asymptomatic and symptomatic EBV infection in the study patients stratified by the type of immunosuppression did not revealed significant differences (Table 1). DISCUSSION
EBV infection is common after pediatric LTx and is associated with the risk of PTLD. Despite the identification of risk factors that predispose patients to develop EBVrelated diseases, a recent review emphasized the limitation of our knowledge of its pathogenesis, variable criteria for establishing the diagnosis, and lack of studies addressing the prevention and treatment of EBV-related diseases.9 Early diagnosis and decreased immunosuppression are important steps in the management of EBV infection and PTLD. Circulating EBV genome level determination has been previously reported to be useful in identifying organ and stem cell transplant recipients at increased risk of PTLD development.3,4 We used an EBV DNA quantitation assay for the early diagnosis of EBV infection and for the presymptomatic treatment of the infection by immunosuppression modulation, using a cut off value of 500 genomes/ 105 PBMC. No patients with EBV DNA levels below 500 genomes/105 PBMC developed EBV infection or PTLD. Those who showed levels of EBV DNA equal to or higher than 500 genomes/105 PBMC were treated by immunosuppression reduction and did not developed symptomatic EBV infection or PTLD. Modulation of immunosuppression was effective in preventing the development of symptomatic infection and caused only in few cases acute rejection episodes (16%), successfully treated by steroid boluses and/or conversion to tacrolimus immunosuppression. Chronic rejection developed in only one child and was not directly related to the treatment of EBV infection. In 13% of patients, symptomatic EBV infection developed in between the scheduled EBV DNA assay. Small children, EBNA negative before LTx, largely composed this group of patients. These data confirm those of other investigators who observed that young transplant patients undergoing primary EBV infection are at higher risk of developing EBV-related diseases,4 and suggest that EBV DNA levels should be assessed more frequently than every 3 months. PTLD incidence in our series was 1%, corresponding to the lower reported ranges.1 A higher risk of PTLD development has been reported in patients receiving tacrolimus immunosuppression.2 Our results did not show differences
MONITORING EBV INFECTION
in the incidence of symptomatic EBV infection clustering the patients according to the immunosuppression therapy. Even if the follow-up time of the study is limited, we can conclude that the use of EBV DNA quantitation in the peripheral blood of pediatric liver recipient seemed to have a positive impact in the early identification of EBV infection, allowing to reduce immunosuppression before the onset of symptomatic EBV infection and PTLD. REFERENCES 1. Newell KA, Alonso EM, Whitington PF, et al: Transplantation 62:370, 1996 2. Younes BS, McDiarmid SV, Martin MG, et al: Transplantation 70:94, 2000
1837 3. Riddler SA, Breinig MC, McKnight JLC: Blood 84:972, 1994 4. Krieger NR, Martized OM, Krams SM, et al: Liver Transpl 6:62, 2000 5. Spada M, Gridelli B, Colledan M, et al: Liver Transpl 6:415, 2000 6. Gerna G, Furione M, Baldanti F, et al: J Clin Microbiol 32:2709, 1994 7. Baldanti F, Grossi P, Furione M, et al: J Clin Microbiol 38:613, 2000 8. Frizzera WP, Hanto DW, Gajl-Peczalska KJ, et al: Cancer Res 41:4262, 1981 9. Paya C, Fung JJ, Nalesnik M, et al: Transplantation 10:1517, 1999