ALBERT

Description: Abstract 3505 Paroxysmal nocturnal hemoglobinuria (PNH) is a non-malignant clonal disorder of hematopoietic stem cells characterized by erythrocyte susceptibility to complement-mediated lysis. Untreated, patients with PNH have a reduced life expectancy compared with age-matched controls. The development of cytopenias with infections related to neutropenia, bleeding associated with thrombocytopenia, and vascular thrombosis all contribute to morbidity and mortality. We previously reported that engrafting donor T-cells mediating graft-vs-host hematopoietic effects can eradicate PNH following reduced intensity (RIC) allogeneic hematopoietic cell transplantation (HCT). Here we present long term follow up data on 16 patients with severe PNH who received a RIC peripheral blood HCT at the NHLBI from May 1999 through January 2007. Eligibility for transplantation included a diagnosis of PNH associated with one or more of the following: 1) Transfusion dependence 2) Prior thrombotic episodes 3) Recurrent debilitating hemolytic crisis. Marrow failure, personal preference or unavailability of the drug precluded the use of eculizumab in all patients. Patients received a T-cell replete G-CSF mobilized blood stem cell transplant from an HLA-matched related donor following conditioning with cyclophosphamide (120mg/kg) and fludarabine (125mg/m2). Patients with a significant transfusion history had equine ATG (40mg/kg/day × 4) added to the conditioning regimen (n= 14). The median CD34+ cell dose was 6.9 × 106cells/kg (range 3.1 to 21.1 × 106) and the median CD3+ cell dose was 2.5 × 108cells/kg (range 1.4 to 4.3 × 108). CSA given alone (n=1) or in combination with either MMF (n=4) or mini-dose methotrexate (n=11) was used as GVHD prophylaxis. FACS analysis on blood samples was performed on all patients at baseline and at multiples time-points post-transplant to measure the percentage of GPI negative PNH-type neutrophils (CD15+/CD66b-/CD16-); the median % of GPI-anchored-protein negative neutrophils pre-transplant was 80.1% (range 5.5%-99%). Half of the patients (n=8) were HLA alloimmunized prior to transplantation with a median 76.5% PRA (range 45%-100%). Neutrophil and platelet recovery occurred at a median 14 days (range 9 to 18) and 12 days (range 5 to 15), respectively. Chimerism assessed in T-cell (CD3) and myeloid (CD14,15) lineages by PCR of short tandem repeats (STR) revealed sustained donor engraftment occurred in both myeloid and T-cell lineages in all patients; the median time to achievement of full donor (〉= 95%) myeloid and T-cell chimerism was 15 and 30 days respectively. FACS analysis on blood samples collected sequentially after transplant revealed evidence for donor immune-mediated eradication of PNH; although GPI-anchored-protein negative neutrophils were detectable early after transplant, over time these populations declined in size and eventually disappeared in all patients at a median 100 days (range 30 to 150) post transplant. Two patients died, one from complications related to acute GVHD (day 169) and one from complications related to a peptic ulcer disease (2.8 years). The cumulative incidence of grade 2–4 acute GVHD was 50% (n=8) and the cumulative incidence of chronic GVHD was 68.8% (n=10). With a median follow-up of nearly 6 years (range 2.6–11 years), 14 patients (87.1%) survive without any evidence of PNH, transfusion independent and off all anticoagulation. Conclusion: Graft-vs-PNH effects that occur after RIC allogeneic HCT result in durable remission and can achieve excellent long-term survival in patients with debilitating PNH. Allogeneic HCT using RIC should be considered a viable treatment option for PNH patients who have failed or are not candidates for eculizumab treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 517 Heavily transfused and alloimmunized patients with bone marrow failure syndromes including aplastic anemia have an increased risk of graft rejection following conventional allogeneic bone marrow transplantation. Results from pilot trials suggest the addition of fludarabine to the conditioning regimen reduces the risk of graft rejection in patients at high risk for this complication. Here we report the results of a fludarabine-based transplant approach in 56 patients with severe aplastic anemia (SAA) or other bone marrow failure syndromes (SAA n=31, MDS-RA n=6, PNH n=16, PRCA n=2, DBA n=1) who were transplanted from May 1999 to November 2008 at the NHLBI. Forty one percent of patients were found to be alloimmunized (median 82% PRA) prior to transplantation as a consequence of prior transfusions. Seventy three percent of patients had received antibody-based immunesuppressive therapy at a median of 303 days (range 21 to 2588) prior to transplantation (horse-ATG n=34, rabbit-ATG n=5, alemtuzumab n=1 and daclizumab n=1). Conditioning with fludarabine (25 mg/m2 × 5 days), ATG (40mg/kg × 4 days) and cyclophosphamide (60mg/kg × 2 days) was followed by infusion of an un-manipulated G-CSF mobilized allograft from an HLA matched (n=52) or single antigen mismatched (n=4) relative. GVHD prophylaxis consisted of cyclosporine (CSA) either alone (n=2) or combined with mycophenolate mofetil (n=10) or mini-dose methotrexate (n=44). The median CD34+ cell dose was 6.6 × 106 cells/kg (range 1.7 to 21.1 × 106 cells/kg) and the median CD3+ cell dose was 2.6 × 108 cells/kg (range 0.5 to 6.9 x108 cells/kg). Nearly half (46 %) of patients received an ABO incompatible allograft (major mismatch n=15; minor mismatch n=11). Despite a high prevalence of pre-transplant alloimmunization, graft rejection and/or graft failure did not occur, with all patients achieving sustained donor engraftment in both myeloid and T-cell lineages. The median time to achievement of full donor (〉= 95%) myeloid and T-cell chimerism was 15 and 30 days respectively. Neutrophil and platelet recovery occurred at a median 15 (range 6 to 24) and 12 (range 5 to 168) days respectively. Major ABO incompatibility was associated with delayed donor erythropoiesis; reticulocyte recovery (〉 60 K/μ L on two occasions) occurred at a median 17 days in those without major ABO incompatibility and 42 days in the recipients of a major ABO mismatched graft, where clearance anti-donor isohemagglutinins was delayed a median 171 days following transplantation. CMV reactivation occurred in 31/50 (62%) patients at risk although no patients died from CMV related mortality. With a median follow-up of 4.5 years (range 1.8–11 years) in surviving patients, overall survival was 87.1%. There were 5 treatment related deaths with two attributable to steroid refractory acute GVHD and one attributable to extensive chronic GVHD. The cumulative incidence of Grade II-IV, III-IV and steroid refractory acute GVHD was 51.8%, 30.4% and 21.4% respectively. The cumulative incidence of chronic GVHD was 72% (23.2% limited and 48.9% extensive), with 42.5% who developed cGVHD having resolution of symptoms allowing discontinuation of systemic immunosuppressive therapy. Conclusion: Fludarabine-based allogeneic peripheral blood stem cell transplantation achieves excellent donor engraftment and long-term disease free survival in heavily transfused and alloimmunized patients with ATG refractory SAA and other nonmalignant hematological disorders associated with bone marrow failure. Efforts to reduce the high incidence of GVHD associated with this approach without increasing the risk of graft failure by manipulating the cellular content of the allograft are currently being explored. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4427 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell progenitors. SAA is treated with immunosuppression or allogeneic stem cell transplantation (SCT), with a successful outcome in a majority. However, 20–40% of patients without a suitable donor for SCT do not respond to immunosuppression and may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Eltrombopag, a small molecule TPO mimetic that binds to mpl, increases platelet counts in healthy subjects, and in patients with chronic immune thrombocytopenic purpura. Both TPO and eltrombopag stimulate more primitive multilineage progenitors and stem cells in vitro. Patients with SAA and thrombocytopenia have very elevated TPO levels; nevertheless, we asked whether pharmacologic doses of eltrombopag could stimulate hematopoiesis in these patients without other options. We are conducting a pilot phase II study of eltrombopag in SAA patients with severe thrombocytopenia refractory to immunosuppressive therapy. Consecutive eligible adult patients were treated with oral eltrombopag at an initial dose of 50 mg daily, with escalation to a maximum dose 150 mg daily, with the goal of maintaining a platelet count of 〉20,000/uL above baseline. Treatment response was measured after three months and was defined as platelet count increases to 20,000/uL above baseline, or stable platelet counts with transfusion-independence for a minimum of 8 weeks. Nine patients have been enrolled and six are evaluable for response to date. Two patients did not respond to treatment. Three patients achieved platelet responses by 12 weeks of treatment, and all have sustained their responses (median follow up 10 months). Four patients exhibited improved hemoglobin levels 12 weeks after starting treatment (median hemoglobin increase of 2.1 g/dL) and two patients who were previously dependent on packed red blood cell transfusions have achieved transfusion-independence. Three neutropenic patients exhibited increased neutrophil counts after treatment with eltrombopag (median increase 0.46K cells/uL). These results provide evidence that eltrombopag can improve platelet counts in patients with severe refractory thrombocytopenia, and perhaps more surprisingly, have a clinically relevant impact on erythropoiesis and myelopoiesis. Updated data will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for thrombocytopenia in refractory severe aplastic anemia patients.

Description: Epstein-Barr virus (EBV) is present in B cells in the blood of healthy people; few studies have looked for EBV in other cell types in blood from patients with lymphoproliferative disorders. We use a new technique combining immunofluorescent cell-surface staining and fluorescent in situ hybridization to quantify both EBV copy number per cell and cell types in blood from patients with high EBV DNA loads. In addition to CD20+ B cells, EBV was present in plasmablast/plasma cells in the blood of 50% of patients, in monocytes or T cells in a small proportion of patients, and in “non-B, non-T, non-monocytes” in 69% of patients. The mean EBV copy number in B cells was significantly higher than in plasmablast/plasma cells. There was no correlation between EBV load and virus copy number per cell. Although we detected CD21, the EBV B-cell receptor, on EBV-infected B cells, we could not detect it on virus-infected T cells. These findings expand the range of cell types infected in the blood. Determining the number of EBV genomes per cell and the type of cells infected in patients with high EBV loads may provide additional prognostic information for the development of EBV lymphoproliferative diseases.