ALBERT

Description: Abstract 4470 Introduction Antibiotics (Abx) have been implicated in immune thrombocytopenia via drug dependent platelet antibodies (DDPA). Our institution has had a DDPA assay available since 1994 which is used to guide clinical decisions. We performed a retrospective review to determine the significance of DDPA. Methods We reviewed the medical records of patients (pts) who tested positive for abx DDPA between 1994 and 2006 and performed a descriptive analysis. Detection of DDPAs was performed using a previously described, modified solid phase red cell adherence assay that detects hapten or immune complex reactions. Results A total of 71 pts were included in this analysis. Multiple classes of abx were tested. Platelet nadir was 1 abx positive for DDPA. Of those with 〉1 abx tested (n=45), 14 (31%) had all tested abx positive. 53% of abx testing took place on or after the abx stop date and 32% of abx tested were administered for

Description: Radioimmunotherapy (RIT) is a useful addition to the armamentarium against NHL. Due to concerns about chronic myelosuppression and the possible inability to mobilize stem cells or tolerate further therapy after RIT, this modality has not been commonly utilized in potential transplant candidates. We report the successful mobilization, collection, transplantation and engraftment of 5 patients with NHL following yttrium-90 ibritumomab tiuxetan. Patients had follicular lymphoma, grade I or II (n=3), transformed follicular lymphoma (n=1) or blastic variant mantle cell lymphoma (n=1). Time from RIT to mobilization was 10 months (median; range 7–27 months). The median number of regimens prior to RIT was 2.5 (range 1–4) including prior autologous transplant (n=1). The median number of regimens following RIT and prior to transplant was 1.5 (range 1–2). All patients exhibited chemosensitive disease. Patients were mobilized with R-ICE/G-CSF (n=4) or G-CSF alone (n=1) and required 2.8 leukaphereses (mean; range 2–5). The CD34 yield was 3.8x106 CD34+ cells/kg (median; range 3.4–5.1x106 CD34+ cells/kg). All patients received busulfan, VP-16, ARA-C and cyclophosphamide (BVAC) as the preparatory regimen. One patient received IL-2 from day 0 to day 11. Time to engraftment was 13 days (median; range 9–19 days) for an absolute neutrophil count (ANC) 〉500 cells/ml and 23 days (median; range 15–44 days) for a platelet count〉20x103/ml. The patient on the IL-2 study showed delayed engraftment of both ANC (day 19) and platelets (day 27). The patient with mantle cell lymphoma, who was undergoing his second autologous transplant, exhibited delayed platelet engraftment (day 44). Time to engraftment did not differ significantly for either ANC (p=0.16) or platelets (p=0.10) in 18 RIT-naïve NHL patients receiving BVAC and an autologous peripheral blood stem cell transplant (PBSC) during the same time period. There were no treatment-related deaths. With a median follow up of 12 months, one patient has died of disease recurrence. Two patients remain in complete remission. Two patients are alive with disease (relapse at 12 months and 14 months) and are currently receiving therapy. No patient has demonstrated laboratory or cytogenetic evidence of a myelodysplastic syndrome. In conclusion, heavily pretreated patients who have received radioimmunotherapy with yttrium-90 ibritumomab tiuxetan and subsequently relapse can successfully undergo mobilization, collection, and autologous PBSC transplant. These patients demonstrate full engraftment, durable remissions and tolerate additional therapy should relapse occur following transplant.

Description: Introduction Crohn's disease and Ataxia Telangiectasia have been managed by extended release of dexamethasone from autologous red blood cells (RBC) with encapsulated dexamethasone sodium phosphate DSP. The EryDexSystem (EDS) is an automated system that loads RBC ex vivo using hypotonic opening of RBC followed by hypertonic resealing of the RBC and washing to prepare the DSP-RBC for infusion. In vivo, DSP is dephosphorylated within the RBC to dexamethasone which passively diffuses into the plasma. The objective of this two-phase study was to elucidate pharmacokinetics (PK) and in vivo 24-hour recovery of RBCs as well as RBC survival (T50) properties of RBC encapsulated DSP. Materials and Methods The study was conduct in two separate Phases, A and B. Phase A, the 24-hour RBC recovery and T50 survival phase, was designed as a randomized, concurrently controlled, single-blind, single-center study to determine the in vivo kinetics of EDS-processed autologous RBC. Healthy volunteer consenting subjects were randomized to receive autologous RBCs prepared using EDS and loaded with either 15-20mg DSP (Group 1A) or sham hypotonic saline (Group 2A). EDS prepared RBC were radiolabeled with 51-Cr following standard methods, and the in vivo labeled RBC followed over 49 days post infusion. The Phase B PK study was designed as an open-label, single-center Phase I study that evaluated two dose levels of DSP encapsulated in RBCs using the EDS. Healthy volunteer consenting subjects were randomized to receive autologous RBCs loaded with either 2.5-5 mg DSP (Group 1B) or 15-20 mg (Group 2B). Post-infusion plasma levels of dexamethasone were followed (over 42 days). Both studies conformed to the Declaration of Helsinki. Results Phase A: Ten subjects (3male; 7 female) were randomized to Groups 1A or 2A. The mean 24-hour RBC recovery ± SD [95% CL] was 77.9 ± 3.3% [73.8-81.9%] and 72.7 ± 10.5% [57.8-85.7%] for Groups 1A and 2A, respectively. The mean ± SD RBC life span in Group 1A was 84.3 ± 8.3 days with a mean T50 of 42.1 ± 4.1 [95% CL: 37.0, 47.3] days, whereas these values were 88.9 ± 6.2 days and 44.4 ± 3.1 [95% CL: 40.6, 48.3] days, respectively, in Group 2A. Sixteen (16) treatment-emergent adverse events (TEAEs) were recorded in Group 1A and 23 in Group 2A. All TEAEs were judged to be unlikely related to the treatment. Phase B: Eighteen subjects (12 male; 6 female) were randomized to Groups 2A and 2B. The actual DSP loading doses (mean ± SEM) were 4.2±0.27 mg and 16.9±0.90 mg. Release of dexamethasone from RBCs in vivo peaked at 1 hour after the end of IV infusion independent of the dose. A detailed summary of the PK parameters for dexamethasone for each treatment group is shown in Table 1. A sustained release of dexamethasone could be detected until 14 and 35 days after the single IV infusion ofEryDex in Group 1B and 2B, respectively. Six (6) TEAEs were reported in each group and were judged to be unlikely related to the study drug or procedure. Conclusion The results for the mean RBC in vivo recovery for DSP-loaded EDS-processed cells meet the FDA criteria for 24-hour RBC recovery of ≥ 75%, without adverse impact on the survival of EDS-processed RBCs. Most of the dexamethasone was rapidly released from the RBCs in vivo with a maximum peak occurring 1 hour after the end of the intravenous infusion, independent of the dose administered, but sustained release of dexamethasone could be detected until 14 and 35 days post infusion for the low and high doses, respectively. DSP-loaded autologous RBCs prepared using the EDS delivered a sustained dose of dexamethasone in vivo. Additional efficacy studies in targeted patient populations are indicated. Disclosures Szczepiorkowski: EryDel S.P.A.: Research Funding. Ferrari:EryDel S.P.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Benatti:EryDel S.P.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Mambrini:EryDel S.P.A.: Employment, Equity Ownership. Hartman:EryDel S.P.A.: Consultancy. Dumont:EryDel S.P.A.: Research Funding.

Description: Autologous stem cell transplant is an effective modality for many patients with lymphoproliferative disorders. Still, the majority of patients with myeloma and many with lymphoma relapse after transplant. Innovative post-transplant immunotherapies are needed. We initiated a phase I immune mobilization trial utilizing dose escalation of IL-2, in combination with GM-CSF and G-CSF, in an attempt to mobilize autologous cytotoxic effector cells, along with peripheral CD34+ hematopoietic progenitor cells. IL-2 began on day 0 of mobilization and continued as a daily SQ injection for 11 days. On day 7 of mobilization, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (day 7–11). On day 11, leukapheresis was performed. Patients then received melphalan (200 mg/m2) followed by reinfusion of cryopreserved autologous peripheral hematopoietic progenitor cells. Phenotypic and functional analyses were performed using peripheral blood mononuclear cells (PBMNC) collected during mobilization on days 0, 7 and 11. Eleven of 12 patients treated to date completed the regimen (myeloma, n=11; NHL, n=1). One patient (NHL) was removed due to progressive disease. The first two dose levels of IL-2 have been well tolerated (dose level 1: 6x105 IU/m2/d; n=6 pts; dose level 2: 1x106 IU/m2/d; n=6 pts) with no ≥grade 3 toxicities noted. Dose escalation of IL-2 continues since the MTD has not been reached. Phenotypic analyses of PBMNC demonstrate an increase in CD3+CD8+ T cells from 17.5% (day 0 baseline) to 22.7% (day 11; p = 0.01). CD56+ NK cells increased from 18.9% (day 0) to 36.0% (day 11; p = 0.002), and CD8+CD56+ NKT cells increased from 8.2% (day 0) to 18.0% (day 11; p = 0.01). Cytotoxicity directed against a human myeloma cell line using peripheral blood lymphocytes was 8.6% at baseline and increased to 43% on day 11 of mobilization (p=0.03). All patients successfully mobilized and received an autologous transplant without delay in engraftment (ANC recovery: day 13 median; range 10–14 days; platelet recovery: day 12 median; range of 0–13 days). These results are encouraging and demonstrate effective mobilization with minimal toxicity and marked in vivo immunomodulatory effects. In addition, the enhanced cytotoxic effector cell number and killing of myeloma cells is quite promising, with additional follow up ongoing to determine the potential impact on survival.

Description: Abstract 4733 Autologous stem cell transplant for myeloma improves survival, but the immunologic mechanisms accounting for this improvement are unknown. Our data indicate that NKG2D+CD8+T cells may be one reason for this benefit. NKG2D, one of four NK cell activating receptors, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. We identified methods for ex vivo expansion of cyclophosphamide – mobilized blood progenitor cells (BPCs) from myeloma patients, as part of a clinical trial (Cytotherapy 2008). Mobilized BPC are cultured in serum-free media with IL-2 (50 IU/ml) and OKT3 (50ng/ml). After 7 days, the CD8+ T cells are isolated, evaluated and tested. Myeloma cells from patients' marrow aspirates are used as targets in cytotoxicity assays. Phenotypic analysis of the expanded cells and the patients' primary myeloma cells was performed using flow cytometry. Ex vivo expansion of cyclophosphamide-mobilized BPCs induces CD8+ T cells that acquire the NKG2D receptor during ex vivo expansion (P 〈 0.03). Three of the 6 known NKG2D ligands are strongly expressed on patients' myeloma cells, including MICA, ULBP1 and ULBP3. Using cytotoxicity assays, autologous effector cells recognize and kill the patient's autologous tumor cells (E:T 50:1, P〈 0.001). When using K562 leukemia cells as targets (lack MHC class I), the NKG2D+CD8+ T cells kill K562, supporting an MHC class I-independent mechanism of tumor cell killing. Blocking the NKG2D receptor on CD8+ T cells prevents killing of autologous myeloma cells (P 〈 0.009). NKG2D-mediated cytotoxicity correlates with the amount of ligand expression on the target. The NKG2D receptor on CD8+T cells recognizes ligands expressed on autologous myeloma cells. The NKG2D+CD8+T cells aggressively kill myeloma cells in a non–MHC restricted and TCR-independent manner. Blocking NKG2D on CD8+ T cells prevents killing of autologous myeloma cells. Since tumor cells often down regulate MHC Class I expression, the ability of NKG2D+CD8+ T cells to recognize and kill autologous myeloma cells in a non-MHC restricted manner may contribute to the beneficial effects in the treatment of myeloma. Ongoing experiments are testing these methods in mouse models and defining the molecular mechanisms involved. Disclosures: Szczepiorkowski: Cersus Corporation: Research Funding; CaridianBCT: Research Funding; BASF: Research Funding; Terumo Corporation: Research Funding; Fenwel, Inc - Scientific Advisory Board: Research Funding. Meehan:Berlex Pharmaceutical: Research Funding.

Description: We initiated an immune mobilization trial in an attempt to mobilize cytotoxic effector cells, along with CD34+ hematopoietic progenitor cells. A Prospective Phase I trial was initiated using dose escalation of IL-2, in combination with GM-CSF and G-CSF. IL-2 began on Day 0 and continued as a daily SQ injection for 11 days. On Day 7, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (Days 7–11). On Day 11, leukapheresis was started if the peripheral blood CD34 + cell count was 〉 5 cells/mcl. The endpoint of collection was ≥ 3 × 106 CD34+ cells/kg. After collection, patients received melphalan (200 mg/m2) followed by infusion of cryopreserved stem cells. Post-transplant GM-CSF began on Day +5 and terminated once the ANC reached 5000 cells/mcl. To date, 9 patients have been treated (myeloma, n = 8; NHL, n = 1) and 7 patients are evaluable. Six patients received IL-2 at Dose Level 1 (6 × 105 IU/m2/d). The remaining 3 patients received IL-2 at Dose Level 2 (1 × 106 IU/m2/d). The MTD of IL-2 has not been reached. One patient (NHL) was removed from the study due to progressive disease. The remaining 8 patients completed the regimen. Toxicities have been mild and have included Grade 2 fever (n=1) on Dose Level 2. All patients were successfully mobilized. The median number of CD34+ cells/kg and MNC/kg collected were 3.4 × 106 (range 2.8 – 4.4 × 106/kg) and 9.5 × 108 (range 0.4 – 1.7 × 109), respectively. Two large volume leukaphereses were required (median; range 1 – 3). Following transplant, the ANC recovered on Day 13 (median; range: 10 – 14 d) and platelets recovered on Day 12 (median; range 0 – 13 d). These preliminary results demonstrate that immune mobilization and collection of an adequate number of hematopoietic progenitor cells is feasible without suppression of hematopoiesis. Toxicities are minimal but the MTD of IL-2 has not yet been reached. Post-transplant engraftment is not delayed. As patient accrual continues, we are currently evaluating the qualitative and quantitative components of the collected cells, including Th1 vs. Th2 cells and the types of dendritic cells mobilized.

Description: A noninferiority study was performed comparing low-dose and standard-dose prophylactic platelet transfusions. A double-blind randomized controlled trial (RCT) was performed in 6 sites in 3 countries. Thrombocytopenic adults requiring prophylactic platelet transfusion were randomly allocated to standard-dose (300-600 × 109 platelets/product) or low-dose (150- 〈 300 × 109 platelets/product) platelets. The primary outcome (World Health Organization [WHO] bleeding ≥ grade 2) was assessed daily through clinical examination, patient interview, and chart review. A WHO grade was assigned through adjudication. The Data Safety Monitoring Board stopped the study because the difference in the grade 4 bleeding reached the prespecified threshold of 5%. At this time, 129 patients had been randomized and 119 patients were included in the analysis (58 low dose; 61 standard dose). Three patients in the low-dose arm (5.2%) had grade 4 bleeds compared with none in the standard-dose arm. WHO bleeding grade 2 or higher was 49.2% (30/61) in the standard-dose arm and 51.7% (30/58) in the low-dose group (relative risk [RR], 1.052; 95% confidence interval [CI], 0.737-1.502). A higher rate of grade 4 bleeding in patients receiving low-dose prophylactic platelet transfusions resulted in this RCT being stopped. Whether this finding was due to chance or represents a real difference requires further investigation. These clinical studies are registered on http://www.clinicaltrials.gov as NCT00420914.