Delay in B-lymphocyte recovery and function following ... - Nature

Bone Marrow Transplantation (2009) 43, 679–684 & 2009 Macmillan Publishers Limited All rights reserved 0268-3369/09 $32.00

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ORIGINAL ARTICLE

Delay in B-lymphocyte recovery and function following rituximab for EBV-associated lymphoproliferative disease early post-allogeneic hematopoietic SCT K Masjosthusmann1,2, K Ehlert1, BR Eing3, J Roth2, G Koehler4, H Juergens1, M Fruehwald1 and AH Groll1 1

Department of Pediatric Hematology and Oncology, University Children’s Hospital Muenster, Muenster, Germany; 2Department of General Pediatrics, University Children’s Hospital Muenster, Muenster, Germany; 3Department of Medical Microbiology, University Hospital Muenster, Muenster, Germany and 4Department of Surgical Pathology, University Hospital Muenster, Muenster, Germany

Treatment with rituximab is highly effective for EBVassociated post transplant lymphoproliferative disease. However, little is known about its immunological sequelae in pediatric allogeneic hematopoietic SCT (HSCT). Time to normal CD19 þ B-lymphocyte values in blood and intravenous immunoglobulin (IVIG) substitution needed to maintain an IgG4400 mg per 100 ml in six consecutive pediatric allogeneic HSCT patients treated with rituximab for symptomatic EBV reactivation were compared with a matched cohort of non-rituximab-treated patients. Follow-up of the six patients ranged from 149 to 1546 days; all but one survived. The mean (±s.d.) time to recovery of CD19 þ B-lymphocytes was 353±142 days as compared with 139±42 in the controls (Po0.01). Similarly, substitution of IVIG as a measure of functional B-cell recovery was extended from a mean of 122±45 to a mean of 647±320 days, and the cumulative dose of IVIG increased from a mean of 1.86±0.51 to 4.4±0.97 g/kg, respectively (Po0.05). One patient had functional B-lymphocyte deficiency for 43 years and ultimately required two stem cell boosts. Rituximab is a live-saving treatment for pediatric HSCT patients but may lead to prolonged and even persistent B-cell deficiency. Bone Marrow Transplantation (2009) 43, 679–684; doi:10.1038/bmt.2008.385; published online 24 November 2008 Keywords: rituximab; transplantation; lymphoproliferative disease; EBV; B lymphocytes; Igs

Correspondence: Professor AH Groll, Infectious Disease Research Program, Department of Pediatric Hematology/Oncology, Children’s University Hospital, Albert-Schweitzer-Strasse 33, Muenster 48129, Germany. E-mail: [email protected] Received 14 January 2008; revised 19 August 2008; accepted 22 August 2008; published online 24 November 2008

Introduction Early-onset EBV-associated post transplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality following allogeneic hematopoietic SCT (HSCT), particular the in unrelated/mismatched patient– donor setting, following in vitro T-cell depletion of the graft and pre-treatment of the recipient with antithymocyte globulin or anti-CD3 MoAb.1,2 The frequency of PTLD in these settings may reach up to 25% of patients with a median time of onset of about 2 months after stem cell reinfusion and an estimated historic mortality of about 80% with conventional therapies, which include reduction in immunosuppression, cytotoxic chemotherapy and use of antiviral agents.1,3 Rituximab is a chimeric MoAb directed against the CD20 antigen found on the surface of normal pre-B and mature B lymphocytes and on 490% of B-cell nonHodgkin’s lymphomas.4,5 Although not approved for this indication, cumulative evidence from phase II clinical trials and case series strongly supports the therapeutic and preemptive benefit of rituximab in the management of PTLD with a striking reversal of the once dismal prognosis.3,6–10 Little is known, however, about the immunological consequences when rituximab is used early after HSCT. Initial investigations in patients with non-Hodgkin’s lymphomas have shown prolonged depletion of CD19 þ lymphocytes in the majority and hypogammaglobulinemia in a considerable fraction of patients.11,12 Here, we report quantitative and functional data of six pediatric patients who received rituximab early post-HSCT for control of EBV infection using B-lymphocyte counts and Ig substitution as markers of B-lymphocyte recovery.

Patients and methods Study design and entry criteria The study was a single-center, retrospective cohort analysis conducted at the Center for Bone Marrow Transplantation

Delay in B-lymphocyte recovery following rituximab early post-HSCT K Masjosthusmann et al

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of the University Hospital of Muenster. Eligible patients had undergone allogeneic HSCT in the pediatric program of the center, had a history of EBV replication in blood or tissue with or without clinical symptoms or histopathologic evidence for EBV-associated PTLD, respectively, and were required to have received at least one dose of rituximab following stem cell reinfusion. Written informed consent for supportive care measures including treatment for EBV disease and the use of anonymized disease-related patient data for scientific investigations was obtained within the consent procedure for HSCT.

Patient enrollment and monitoring Patient enrollment was from 05/2003 until 08/2006, and follow-up was at least until day þ 360 after stem cell transfusion in case of patient survival. Data collection was accomplished with a standardized case report form. Patients were monitored with regular clinical examination, laboratory monitoring and imaging studies as provided by institutional policies. During hospitalization, patients underwent daily clinical and biochemical assessment. Following discharge, patients were followed up at least once weekly until day þ 100, at least every other week between days þ 100 and þ 180, and as dictated by clinical status thereafter. Complete clinical and laboratory workup were performed on each occasion, and further diagnostic studies were performed as clinically indicated. Diagnosis of EBV replication and disease The diagnosis of B-cell PTLD was based on clinical, morphologic, immunophenotypical and molecular genetic analysis and classified according to the World Health Organization classification.13 EBV replication was screened for twice weekly during hospitalization and at least once weekly in the outpatient setting by means of a pan-herpes virus PCR assay (Herpes Consensus Generic, Argene Biosoft, Varhiles, France).14 In case of a positive signal or for follow-up of established EBV replication, EBV load was measured and monitored at similar intervals in PBMCs and plasma samples by means of a real-time quantitative PCR (artus EBV RG PCR, Qiagen, Hilden, Germany) as described in detail elsewhere.15,16 The results were expressed as the number of copies per microgram of DNA for PBMCs and as the number of copies per milliliter of plasma, respectively. Rituximab treatment and response evaluation Rituximab was administered at a dose of 375 mg/m2 once a week by i.v. infusion at the rate recommended by the manufacturer, following premedication with paracetamol and antihistamins. Treatment was continued until a satisfactory clinical/molecular response or occurrence of a treatment limiting adverse event. Responses to treatment were assessed using clinical symptoms and findings, imaging studies guided by clinical findings, and the time course of EBV replication in blood. Clinical responses were evaluated as complete, partial, stable or progressive disease as defined previously.2,17 Bone Marrow Transplantation

Assessment of B-lymphocyte recovery and function The end points of the study included the time from stem cell transfusion to normal CD19 þ B-lymphocyte values in blood and both the time and the cumulative dose of intravenous immunoglobulins (IVIGs) needed to maintain an IgG value above 400 mg per 100 ml as compared with a matched cohort of non-rituximab-treated patients without PTLD who were scheduled to receive Ig substitution per protocol at 0.2 g/kg twice per month until at least day þ 180 post transplant. The minimum required time of follow-up for inclusion into the statistical analysis was þ 365 days post transplant. Analyses of lymphocyte subpopulations and serum IgG were performed at baseline and at least monthly thereafter. Analysis of lymphocyte subsets was performed by FACS using a Sorter Vantage SE Analyzer (Becton Dickenson, Franklin Lakes, NJ, USA) and a panel of two-colour fluorescence marker CD19 PerCP/CD3 Cy5.5 antibodies (Becton Dickenson). Serum IgG concentrations were measured by an anti-Human-IgG assay (Behring, Germany) and an immunonephelometer (BN II; Behring, Germany). Published age-corrected ranges of normal values served as reference.18,19 Owing to the limited sample size of the population under study, matching of the control cohort was 1:1 and limited to underlying disease, gender and age. Statistical considerations Data were analyzed by descriptive statistics if not indicated otherwise. For statistical comparisons of continuous data, the Mann–Whitney U-test was used.

Results Six of 101 patients (5.9%) who underwent allogeneic HSCT in the pediatric program of the Center for Bone Marrow Transplantation between July 1999 and June 2006 developed symptomatic EBV reactivation in blood or tissue and received treatment with rituximab.

Demographics and transplant characteristics Patient demographics and transplant characteristics are summarized in Table 1. Four of the six patients were female; the median age was 12.5 years (range, 1 to 18). Underlying conditions included AML in first remission (AML; 3), myelodysplastic syndrome/refractory cytopenia (MDS-RC; 2) and mucopolysaccharidosis (MPS; 1). Three patients had undergone myeloablative and three reducedintensity conditioning regimens; five patients were transplanted from matched unrelated donors following lymphocyte depletion in vivo, and one was transplanted from a matched family donor. Standard immunosuppression consisted of CsA given from day 1 until day þ 100 ( matched family donor) or þ 180 (matched unrelated donors), and two to four doses of MTX (10 or 15 mg/m2 q.d.), administered on days þ 1, þ 3, þ 6, þ 11. Primary engraftment was achieved in all patients and occurred after a median of 22.5 days (range, 16–28); two patients had secondary graft failure at days 98 and 225 (patient nos. 2 and 4). Four patients developed acute grades I to IV


681 Table 1 Patient number 1 2 3 4 5 6

Patient demographics and transplant characteristics of the six patients with B-PTLD Age (years)

Gender

Underlying condition

Status at HSCT

Donor type a

Conditioning regimen

1 14 11 18 4 16

M M F F F F

MPS-1 SAML AML MDS-RC AML MDS-RC

NA CR-1 CR-1 NA CR-1 NA

MUD MUD MUD MUD MRD MUD

FAMP/MEL/TLI ALEMTUZUMAB BU/CY/MEL/ATG BU/CY/ATG FAMP/TT/ATG BU/CY FAMP/TT/ATG

Engraftment (days post-HSCT)

Acute GVHD (grade)

Steroids (days)

25 17 16 24 28 21

I III — II IV —

44 55 — 33 192 —

Abbreviations: ATG ¼ antithymocyte globulin; CR-1 ¼ complete first hematological remission; FAMP ¼ fludarabine monophosphate; HSCT ¼ hematopoietic SCT; MDS-RC ¼ myelodysplastic Syndrome-refractory cytopenia; MEL ¼ melphalane; MPS-1 ¼ mucopolysaccharidosis type 1; MRD ¼ matched related donor; MUD ¼ matched unrelated donor; NA ¼ not applicable; PTLD ¼ post transplant lymphoproliferative disease; sAML ¼ secondary AML; TLI ¼ total lymph node irradiation; TT ¼ thiotepa. a All but one patient (no. 6; PBSCs) had received BM as source of hematopoietic progenitors.

Table 2

Diagnosis of PTLD, details of rituximab therapy, and outcome of the six patients with B-PTLD

Patient number

PTLD type

Site(s) involved a

1 2 3 4 5 6

B-PTLD B-PTLD B-PTLD B-PTLD B-PTLD B-PTLD

Blood, LNsa Blood Blood Blood, guta Lunga Blood, LNsa

Diagnosis (days postHSCT)b

Maximum viral load (copies/ml)

Rituximab cumulative (mg/msqu)

Time to negative viral load (days)c

PTLD outcome

41 129 75 97 182 36

59777 1513 11021 10614 NA 1000

1500 750 375 1500 375 375

92 19 9 31 NA 12

CR CR CR CR CR CR

Hematologic outcome

Last follow-up (days post-HSCT)

Alive in CR Death (day +149) Alive in CR Alive in CR Alive in CR Alive with hypogamma-globulinemia

811 149 538 594 1206 1546

Abbreviations: HSCT ¼ hematopoietic SCT; LN=lymph nodes; NA ¼ not applicable; PTLD ¼ post transplant lymphoproliferative disease. a Biopsy proven. b The day of diagnosis was defined as the day when treatment was initiated. c Viral load in plasma as determined by quantitative PCR.

GVHD and received therapeutic dosages of glucocorticosteroids (initial dosage: 1 mg/kg methylprednisolone b.i.d.) for 30 to 192 days. None of the patients developed chronic GVHD.

Diagnosis of PTLD, rituximab therapy and outcome Details of PTLD diagnosis, rituximab therapy and patient outcome are shown in Table 2. Three patients (patient nos. 1, 4 and 6) had biopsy proven tumoral involvement with raised EBV load in blood and one patient (patient no. 5) had pneumonia associated with EBV replication in lung tissue and deep respiratory secretions; the other two patients had refractory fever associated with raised EBV load (infectious mononucleosislike PTLD). The median maximum viral load in plasma was 10614 copies/ml (range, 1000–59777 copies/ml). Treatment with rituximab was commenced at diagnosis at a median of day þ 86 (range, þ 36 to þ 182 days) after stem cell transfusion. The mean cumulative dose of rituximab was 812 mg/msqu (range, 375–1500); three patients received one single infusion, one patient two infusions and two patients received four infusions. Rituximab was well tolerated in all patients without discontinuations because of adverse events. All patients achieved a complete response following treatment with rituximab without breakthrough or reactivation; the median time to disappearance of detectable viral replication in blood was 19 days (range, 9–92 days) in the five patients with EBV detected in blood.

At a median follow-up of þ 702 days after stem cell transfusion (range, 538–1546), five patients were alive and in continuing hematological remission. The remaining patient (patient no. 2), whose course post transplant had been additionally complicated by disseminated adenovirus infection, died with cleared EBV replication from secondary refractory graft failure on day þ 150 with sepsis syndrome and probable pulmonary aspergillosis.

B-lymphocyte recovery and function B-lymphocyte recovery and function are depicted in Figure 1. The mean (±s.d.) time to recovery of CD19 þ lymphocytes in rituximab-treated patients to age-corrected normal values was 353±142 days (median: 304; range: 213–578 days) post transplant and significantly prolonged as compared to the control cohort (139±42 days (median: 138; range: 89–187 days; P ¼ 0.0079 by Mann–Whitney U-test). Similarly, substitution of IVIG as a measure of functional B-cell recovery was extended from a mean of 122±45 (median: 105; range: 74–187 days) in the control cohort to a mean of 647±320 days (median: 550; range, 356–1133 days; P ¼ 0.0159), and the cumulative dose of IVIG required to maintain an IgG value of 4400 mg per 100 ml increased from a mean of 1.86±0.51 (median: 1.7; range: 1.3–2.5 g/kg) to 4.4±0.97 g/kg (median: 4.9; range: 3–5 g/kg), respectively (P ¼ 0.0155). There was no apparent relationship between quantitative and qualitative B-lymphocyte recovery and the cumulative dose of rituximab Bone Marrow Transplantation


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Time to CD19+ recovery [days ±SD]

a

600

450

300

150

0 Rituximab

Controls

Duration of IVIG substitution [days ±SD]

b 1200 1000

800 600

400

200

0 Rituximab

Controls

Figure 1

B-cell recovery and function in rituximab-treated patients and in matched controls. (a) Mean±s.d. time to normal CD19 þ lymphocytes in days after stem cell transfusion; Po0.01 by Mann–Whitney U-test for the comparison of rituximab-treated patients with the control cohort. (b) Mean±s.d. time of intravenous immunoglobulin (IVIG) substitution in days after stem cell transfusion to maintain a serum IgG above 400 mg per 100 ml; Po0.05 by Mann–Whitney U-test for the comparison of rituximabtreated patients with the control cohort. Note that two patients (one in each cohort) did not meet the minimum required time of follow-up of þ 365 days post transplant because of early mortality and were excluded from the analysis. One additional patient (patient no. 3) was excluded from the analysis of Ig recovery because of hypertransfusion with IVIG during the week before rituximab therapy. This patient had an IgG concentration of 2400 mg per 100 ml at baseline, received a total amount of 0.80 g/kg of IVIG during follow-up on two occasions but never reached a serum IgG of o400 mg per 100 ml. Open circles, mean values; closed circles, control patients; closed square, patient no. 1; closed downward arrow, patient no. 3; closed upward arrow, patient no. 4; closed star, patient no. 5; closed hexagone, patient no. 6.

administered. Of note, one patient (patient no. 6) had isolated functional B-cell deficiency with persistent hypogammaglobulinemia lasting for more than 3 years post transplant and ultimately received repeat stem cell boost to achieve immune recovery.

Discussion The results of this retrospective cohort analysis suggest that reduced immunosupresssive therapy and administration of Bone Marrow Transplantation

rituximab is effective for management of early B-PTLD in pediatric allogeneic HSCT recipients, leading to persistent control of EBV disease in all six patients studied without further interventions. However, rituximab treatment led to a significant delay in the recovery of CD19 þ B lymphocytes and in autochthonous IgG production as compared with matched untreated controls and hypogammaglobulinemia in one patient that persisted for more than 3 years duration. On the basis of the antibody’s mechanism of action, depletion of B lymphocytes and hypogammaglobulinemia is an expected adverse event following administration of rituximab.11,12 Among the 166 cancer patients in the pivotal non-Hodgkin’s lymphoma study, circulating CD19 þ lymphocytes were depleted within the first three doses with sustained depletion for up to 6–9 months after treatment in 83% of patients. In addition, there were sustained and statistically significant reductions in both IgM and IgG serum levels observed from 5 through 11 months following rituximab administration, but only 14% of patients had reductions in IgM and/or IgG serum levels below the normal range.12 Lymphoma patients who receive rituximab as adjuvant or maintenance following autologous HSCT, however, have been reported to have an increased risk of developing severe and long-lasting hypogammaglobulinemia despite quantitative CD19 þ B-lymphocyte recovery.20–23 Laboratory studies in patients with lymphoma or alloantibodies before kidney transplantation have shown a delayed recovery of CD19 þ /CD27 þ B-memory cells and impaired isotype expression following rituximab therapy as seen in patients with common variable immunodeficiency, suggesting that rituximab can affect not only B-cell quantities but also the recovery of functional B-cell repertoires and differentiation into plasma cells.20,21,24,25 Little is known of the consequences of rituximab therapy on B-lymphocyte depletion and recovery in patients with PTLD following allogeneic HSCT, particular in pediatric patients. Although incubation of B cells with anti-CD20 antibody depletes normal circulating B cells and has variable effects on cell cycle progression and signaling, the detailed biologic functions of CD20 remain uncertain.26 B-cell lymphopenia of approximately 6 months duration has been observed in 12/15 adult patients who received rituximab in preemptive indication,8 and a few cases of prolonged hypogammaglobulinemia have been published,27–30 of whom one provided laboratory evidence for similar mechanisms as described in the lymphoma setting. However, the situation may be more complex as additional factors may contribute following allogeneic HSCT, including engraftment, expansion and differentiation of lymphopoiesis, immunological processes between host and graft, the action of immunosuppressive agents and active EBV infection, which upon itself may be associated with immunodeficiency31 and secondary graft failure as in two of the six patients described in this report.32 Multiple case studies, retrospective analyses and phase II trials have shown the therapeutic benefit of rituximab in EBV-associated, CD20 þ PTLD following allogeneic HSCT;6–9,33–36 however, the optimal approach to management of PTLD in this population is still evolving and remains to be defined.3,10 As assessed by PCR amplification


683

of EBV-specific DNA in blood, EBV reactivation is common post-allogeneic HSCT, but only a minority of patients with EBV detected in blood during monitoring will progress to overt PTLD.33,35 Quantitative monitoring of EBV replication and preemptive therapy with rituximab only in the fraction of patients with high-level reactivation has been shown to improve outcome as compared with historical controls,8 but this approach may expose approximately half of the so selected patients to unnecessary therapy as they may be salvaged with rituximab when developing clinical signs and symptoms of PTLD.35 An alternative to the approach of ‘prompt treatment’ is the combined use of quantitative real-time PCR monitoring of EBV DNA in blood with monitoring of EBV-specific CD8 þ T cells by peptide-HLA class I tetramers. Failure to detect EBV-specific CD8 þ T cells in patients with highlevel EBV reactivation in blood has been shown by several investigators to be associated with the subsequent development of EBV PTLD.36–38 Thus, simultaneous monitoring of EBV DNA and EBV-specific CD8 þ T cells in blood may serve to more specifically guide individual decisions on preemptive interventions, including modifications in immunosuppression, administration of rituximab, donor lymphocytes and specific cellular therapies that may have new and serious clinical consequences for the individual patient. Rituximab is a potentially live-saving treatment option for patients developing EBV replication and disease early post-HSCT but may result in prolonged and even persistent quantitative and functional B-cell deficiency. The immunological sequelae of rituximab therapy following administration early after HSCT are poorly understood. As many HSCT patients will become long-term survivors, further systematic laboratory investigations and a phase IV clinical program are needed to understand and monitor potential immunological consequences of rituximab therapy in this special population.

5 6

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9

10

11

12

13

14

Acknowledgements We thank Claudia Katerkamp, Hedwig Kolve and Maria Waeltermann for administrative and technical support. The results of this analysis were presented at the 25th annual meeting of the European Society for Paediatric Infectious Diseases, Porto, Portugal, 2–4 May 2007.

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17

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