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Anti-CD20 Monoclonal Antibody (Rituximab) Treatment for Epstein-Barr Virus-associated, B-cell Lymphoproliferative Disease in Pediatric Liver Transplant Recipients

Serinet, Marie Odile*; Jacquemin, Emmanuel*; Habes, Dalila*; Debray, Dominique*; Fabre, Monique; Bernard, Olivier*

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Journal of Pediatric Gastroenterology and Nutrition: April 2002 - Volume 34 - Issue 4 - p 389-393
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Lymphoproliferative disease is a serious complication of liver transplantation in pediatric patients (1). Its incidence is about 4% to 9%, and mortality rates reach 50%. Most cases are Epstein-Barr virus (EBV)–associated B-cell lymphoproliferative disorders. Posttransplant B-cell lymphoproliferative disease (PTLD) types vary, ranging from polyclonal lymphoid proliferation, which mostly occurs early after transplantation when immunosuppression is strong, to true lymphoma, which usually occurs later in the posttransplant period (2). The first therapeutic approach is to stop or reduce immunosuppression (1,3). Sometimes, additional surgical resection for localized tumors or chemotherapy in malignant forms, such as Hodgkin-like or Burkitt-like lymphomas, are indicated (2–4). Adoptive-cell immunotherapy and anti–B-cell immunotherapy have been proposed as alternative therapies and have shown promise in treating PTLD (5–7). Adoptive-cell therapy is not as feasible and may be hampered by the risk of graft versus host disease in solid organ transplantation (5). B-cell–specific monoclonal antibodies (anti-CD21 and anti-CD24) have induced complete remission in some patients with oligoclonal forms and also with severe PTLD after solid or bone marrow transplantation, but these antibodies are no longer available (6,7). More recently, a commercially available anti-CD20 monoclonal antibody (rituximab) has been used with success in adults with PTLD after solid organ transplantation or after bone marrow transplantation (8–12), and has been used in a few pediatric cases, mainly after bone marrow transplantation (13–14). We report the outcome of anti-CD20 antibody therapy for EBV-associated PTLD in six pediatric liver transplant recipients.


Between September 1997 and December 1999, 63 pediatric liver transplantations were performed in our unit. Among the recipients, six children experienced EBV-associated PTLD and had evidence of progressive disseminated disease or bulky lesions. They received anti-CD20 monoclonal antibody. Table 1 describes the main characteristics of recipients and of their PTLD. At the time of transplantation, recipient ages ranged from 8 months to 16 years. Four children were transplanted for biliary atresia, one for Alagille syndrome, and one for progressive familial intrahepatic cholestasis. After transplantation, the immunosuppressive regimen consisted of prednisone, cyclosporine, and, in four children, azathioprine. In addition to prednisone and cyclosporine, patient d received anti–interleukin 2 receptor antibody as part of a combined liver–kidney graft immunosuppression protocol. Prophylactic treatment with acyclovir was given to all children for 3 months after transplantation. In four patients, acute rejection episodes were diagnosed with liver biopsy specimens in the first month after transplantation. Treatment of rejection consisted initially of methylprednisolone pulses. Four patients experienced cortico-resistant acute rejection, and therapy was switched from cyclosporine to tacrolimus. At the time of PTLD diagnosis, cyclosporine blood trough concentration was 226 ng/mL in one patient, and tacrolimus blood trough concentrations ranged from 6 to 15 ng/mL (mean, 11ng/mL) in the other patients.

Recipient and posttransplant B-lymphoproliferative disease characteristics

Posttransplant lymphoproliferative disease was diagnosed 2 to 4 months after transplantation. An extensive search for tumor masses was performed (total body scan, bone marrow aspiration, gastrointestinal endoscopy). Tumors were localized in several organs in two patients and were restricted to the liver or to the gut in three patients. In the sixth patient, no tumor was initially detectable, but PTLD was suspected because of prolonged high fever with signs of multiorgan failure and opportunistic infections associated with a high EBV load and serum immunoglobulin M monoclonal peak. In this patient, histologic proof of PTLD was obtained secondarily from cerebral tissue obtained during Ommaya reservoir placement for intrathecal therapy (see Results). Of the six children, three experienced PTLD after EBV primary infection and three after reactivation. At the time of diagnosis, spontaneous blood B-lymphocyte proliferation and serum monoclonal or oligoclonal immunoglobulin were present in four children. The two remaining patients had no abnormal immunoglobulin component in their sera and no spontaneous blood B-cell proliferation. Biopsies of masses showed a monomorphic or polymorphic B-cell infiltrate expressing CD20 antigen that related to EBV. Epstein-Barr virus was detected by immunohistochemistry with a monoclonal anti–latent membrane protein antibody or by in situ hybridization with a specific Epstein-Barr virus-encoded RNA (EBER) oligonucleotide probe.

In all patients, tacrolimus or cyclosporine therapy was withdrawn and prednisone therapy was continued at 0.5 mg/kg to 2 mg/kg daily. Within 3 to 18 days, anti-CD20 monoclonal antibody (rituximab; Roche Pharma, Neuilly-sur-Seine, France) was given intravenously at 375 mg/m2 once a week for a total of 4 weeks in four children and a total of 3 weeks in two. Parents gave informed consent in all cases.


Treatment Tolerance

During the first infusion of anti-CD20 antibody, headaches occurred in one patient and an anaphylactic reaction in another, probably because of excessive infusion speed. In this patient, symptomatology was reversible after stopping the infusion and did not recur at a slower rate of infusion. All patients have had severe hypogammaglobulinemia, requiring intravenous immunoglobulin supplementation. In the three patients who survived, immunoglobulin supplementation was necessary for a mean period of 19 months to maintain serum gamma-globulin concentration above 5g/L. Two patients experienced transient neutropenia, reversible after recombinant human granulocyte-colony stimulating factor injections.

Treatment Efficacy, Tumoral and Hepatic outcomes

Table 2 shows tumoral and hepatic outcomes. Anti-CD20 antibody infusion was associated with complete remission in the five patients in whom tumoral masses were initially present. Complete remission was defined by clinical and radiologic disappearance of any mass associated with a decrease of EBV load and disappearance of abnormal immunoglobulin components in serum. The disappearance of the masses occurred within 1 to 2.5 months after beginning treatment. As mentioned above, one patient experienced symptoms later with seizures because of a cerebral tumor. However he had systemic remission as evidenced by decreased EBV load and disappearance of serum monoclonal immunoglobulin. As an alternative to chemotherapy and on the basis of a previous report of anti-CD21 monoclonal antibody use (15), intrathecal injections of anti-CD20 antibody were attempted to treat the central nervous system lesion. This approach was chosen to avoid the hepatotoxicity of chemotherapy in a patient with liver injury caused by chronic rejection (see below) (16). The cerebral lesion size increased and polychemotherapy was started, inducing a clearcut decrease in tumor size, but treatment was complicated by liver failure.

Outcome of the six patients with posttransplant B-lymphoproliferative disease after onset of treatment with anti–CD20 antibody

Five children experienced acute liver graft rejection episodes, confirmed by liver histology. Rejection episodes occurred 10 days to 2.5 months after the onset of anti-CD20 antibody treatment. Immunosuppression therapy was then reintroduced, tacrolimus in three children and cyclosporine in two. Three children experienced subsequent chronic rejection.

At last follow-up, three of the six children were alive and in remission, 15, 28, and 36 months after the onset of PTLD, and have normal liver test results. Among the three others, one died of liver failure caused by chronic rejection and one with chronic rejection died of uncontrolled sepsis. Both died despite remission of PTLD 4 months after its onset. The third patient died of liver dysfunction induced by chemotherapy.


Immunotherapy with B-cell–specific monoclonal antibody is an interesting approach to treating EBV-associated PTLD. Indeed, monoclonal antibodies such as anti-CD21 and anti-CD24 have been tested with success for this indication, with moderate toxicity and side effects compared with chemotherapy, but these molecules are no longer available (6,7). Anti-CD20 antibody, as are anti-CD21 and anti-CD24 antibodies, is a monoclonal antibody that specifically binds to the CD20 antigen of normal and malignant B cells, and results in antibody- and complement-dependent cytotoxicity. Unlike anti-CD21 and anti-CD24, it is a mouse–human chimeric antibody that theoretically should be better tolerated and is commercially available. Initially used with encouraging results to treat low grade B-cell lymphomas in relapse (17), more recently this molecule has also proven effective in adult patients with PTLD (8–12). There have been only a few reports of using anti-CD20 antibody in children with EBV-associated PTLD (12–14).

Infusions of anti-CD20 antibody resulted in rapid control of PTLD in all patients but were not effective in preventing or treating central nervous system involvement. This could be expected because monoclonal anti-CD21 and anti-CD24 antibodies do not cross the blood–brain barrier (15). Hepatic evolution after withdrawing immunosuppression and beginning anti-CD20 antibody treatment was complicated by acute rejection that appeared early in most cases. Indeed in three patients, despite resumption of immunosuppression, rejection was extremely severe and refractory, leading eventually to death directly or indirectly. In this series, death was not caused by progression of lymphoproliferative disease, because two children died after tumor remission and one died after the cerebral tumor had decreased dramatically. Under such circumstances, in absence of immunosuppression and with immunotherapy, the host's immune response may return faster, leading to early acute graft rejection that may be more difficult to control and that evolves toward lethal chronic rejection.

Decreased serum EBV load seems to correlate with restoration of cellular immune response, as evidenced by the regression of lymphoproliferative disease and the appearance of hepatic rejection (18). By depleting B cells, rituximab also may favor the decrease of EBV copy number. As shown for patient b in Figure 1, EBV viral load decreased dramatically within the first month of treatment with anti-CD20 antibody. In this patient, resumption of immunosuppression before the end of the first month might have prevented rejection. In such patients, regularly monitoring EBV load may help adapt the immunosuppression treatment under immunotherapy (18,19).

FIG. 1.:
Epstein-Barr virus (EBV) load profile, with anti-CD20 antibody treatment in patient b, measured by semiquantitative polymerase chain reaction and expressed as number of EBV copies per 105 lymphocytes. IS, immunosuppression.

In conclusion, immunotherapy with anti-CD20 monoclonal antibody (rituximab), used as first-line therapy, provides an interesting treatment for children with severe EBV-associated PTLD that occurs early after liver transplantation. Rituximab is associated with moderate side effects and allows rapid control of lymphoproliferative disease, but does not prevent cerebral localization. The rapidity of tumor mass decrease with rituximab therapy should allow rapid resumption of immunosuppression, while still receiving rituximab therapy, or may even allow continued immunosuppression at a low level to prevent potentially lethal liver graft rejection. This report adds to the few in the pediatric literature concerning the use of rituximab in PTLD. A large, multicenter, pediatric study of rituximab for lymphoproliferative disease after liver transplantation is warranted to confirm the efficacy of such therapy. Such a study also may clarify which type of PTLD responds to this therapy.


The authors thank Prof. Denis Devictor and Dr. Philippe Durand (Pediatric Intensive Care Unit, Bicêtre Hospital) for their contribution to patient care, and Prof. Alain Fischer, Prof. Stéphane Blanche, and Dr. Pierre Quartier (Immunology and Hematology, Necker Hospital, Paris, France) for therapeutic advice.


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Lymphoproliferative disease; Epstein-Barr virus; Liver transplantation; Children; Rituximab

© 2002 Lippincott Williams & Wilkins, Inc.