Impact of Pre-Emptive Rituximab Treatment for Epstein-Barr Virus Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation: A Longitudinal Study of 110 Patients

Background: Epstein-Barr Virus (EBV) reactivation and EBV-associated post-transplant lymphoproliferative disease (PTLD) are well recognized complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with a high mortality. Pre-emptive anti-CD20 therapy with Rituximab has proved successful to reduce PTLD incidence in the setting of EBV reactivation. However, since CD20 expression is not confined to the malignant cells, normal B cells are also targeted by Rituximab, which can be of concern in patients who are already immunosuppressed, especially with regard to long-term infectious complications. This study aimed to analyze immune recovery, and to define the incidence of infectious complications and outcomes following Rituximab pre-emptive therapy.

Patients and Methods: One hundred and ten consecutive patients from a single center, who underwent allo-HSCT between 2009 and 2016 and treated with rituximab for EBV reactivation, were included. Based on ECIL recommendations, EBV-DNAemia was routinely monitored using real-time quantitative PCR to quantify EBV genomes. Patients with two consecutive positive results received weekly Rituximab until EBV-DNAemia clearance. Clinical data [infections, overall survival (OS), event-free survival (EFS), non-relapse mortality (NRM), relapse incidence (RI), acute graft-versus host-disease (GVHD) and chronic GVHD] were assessed together with sequential evaluation of the following immunological parameters (T cells, B cells, Ig levels and neutrophil counts). Moreover, in a subset of 38 patients, immune B-cell subset quantification were performed using multi-color flow cytometry at months (M) 1, 3, 6 and 12 after Rituximab administration.

Results: In this cohort, median age was 55 (range, 16-71) years. Diagnoses were myeloid malignancies (71%), lymphoid malignancies (25%) or severe aplastic anemia (4%). Thirty-two percent of patients received a graft from a matched sibling donor, 44% from an unrelated donor, and 24% from a haplo-identical donor. Ninety-three patients received a reduced intensity conditioning regimen based on fludarabine, busulfan +/- thiotepa. Ninety-two percent of the patients received ATG as part of the conditioning regimen. In the haplo subgroup, patients received standard post-transplant cyclophosphamide. All patients received a combination of cyclosporine A and mycophenolate mofetil for GVHD prophylaxis, except for patients with a sibling donor who received cyclosporine A only.

Median times from allo-HSCT to EBV reactivation and from allo-HSCT to rituximab administration were 52 and 60 days, respectively. Median number of Rituximab administrations was 3 (range, 2-8). Five patients developed PTLD despite pre-emptive Rituximab (including one case of PTLD-related death). The cumulative incidence (CI) of overall infections at 2 years was 36% (95%CI: 27-46%). Twenty-one percent developed at least one bacterial infection, 19% a viral infection, and 6% a fungal infection. At 2 years, EFS was 63% (95% CI: 53-73), OS was 69% (95% CI: 60-78), NRM was 18.6% (95% CI: 11-27) and RI 18% (95% CI: 11-27). CI of grade 2-4 acute GVHD was 6.4% (95% CI: 11-27). CI of chronic GVHD 34.5% (95% CI: 3-12) and CI of extensive cGVHD was 9%. After taking into account GVHD, donor lymphocyte infusion, Ig IV substitution and relapse, at M1, M3, M6, M12, the median lymphocytes counts were 0.62/µL, 0.7/µL, 0.88/µL and 1.62/µL, and Ig levels 6.3g/L, 6g/L, 6.1g/L and 6.6g/L, respectively. Twenty-three percent of patients developed a neutropenia that was considered to be related to Rituximab administration. A slow B cell reconstitution was observed with median counts of 0.12/µL at M1, 0.55/µL at M3, 0.70/µL at M6 and 0.90/µL at M12, with over time a decreased proportion of naïve B cells (CD24 low, CD27-, IgD+, CD38 low) contrasting with an increased proportion in transitional B cells (CD24+, CD27-, IgD+, CD38+).

Conclusion: Pre-emptive Rituximab treatment for EBV reactivation after HSCT is associated with high infection rate and delayed immunological reconstitution, with a quarter of the patients developing Rituximab-related neutropenia. However, these unfavorable effects did not negatively impact general outcomes, and the relatively very low CI of extensive cGVHD may suggest a favorable impact of anti-CD20 therapy on this late complication.