ALBERT

Description: Allogeneic stem cell transplantation with umbilical cord blood (UCB) cells is limited by the cell dose a single unit provides recipients. Ex vivo expansion is one strategy to increase the number of cells available for transplantation. Aastrom Biosciences developed an automated continuous perfusion culture device for expansion of hematopoietic stem cells (HSCs). Cells are expanded in media supplemented with fetal bovine serum, horse serum, PIXY321, flt-3 ligand, and erythropoietin. We performed a phase 1 trial augmenting conventional UCB transplants with ex vivo–expanded cells. The 28 patients were enrolled on the trial between October 8, 1997 and September 30, 1998. UCB cells were expanded in the device, then administered as a boost to the conventional graft on posttransplantation day 12. While expansion of total cells and colony-forming units (CFUs) occurred in all cases, the magnitude of expansion varied considerably. The median fold increase was 2.4 (range, 1.0-8.5) in nucleated cells, 82 (range, 4.6-266.4) in CFU granulocyte-macrophages, and 0.5 (range, 0.09-2.45) in CD34+ lineage negative (lin–) cells. CD3+ cells did not expand under these conditions. Clinical-scale ex vivo expansion of UCB is feasible, and the administration of ex vivo–expanded cells is well tolerated. Augmentation of UCB transplants with ex vivo–expanded cells did not alter the time to myeloid, erythroid, or platelet engraftment in 21 evaluable patients. Recipients of ex vivo–expanded cells continue to have durable engraftment with a median follow-up of 47 months (range, 41-51 months). A randomized phase 2 study will determine whether augmenting UCB transplants with ex vivo–expanded UCB cells is beneficial.

Description: We have identified a small molecular weight compound, SCH 14988, which specifically stimulates in vitro granulocyte-colony stimulating factor (G-CSF ) production from activated human peripheral blood mononuclear cells and monocytes but not other cytokines or CSFs with hematoregulatory activity. In vivo administration of SCH 14988 to mice rendered neutropenic by cyclophosphamide treatment resulted in the accelerated recovery of the peripheral neutrophil compartment. This activity correlated with increased in vivo G-CSF levels and stimulation of marrow granulopoiesis, and was comparable to that of exogenously administered recombinant human G-CSF. No alterations to other leukocyte populations in peripheral blood, spleen, or the peritoneal cavity were observed. These findings suggest that SCH 14988 may be clinically useful to enhance neutrophil granulopoiesis, as well as to study the mechanisms involved in G-CSF gene regulation.

Description: Careful planning is critical to ensure blood availability during times of reduced supply and/or increased demand, such as infectious pandemic, act of terrorism or natural disaster. Possible strategies include triaging of supply to prioritize support where transfusion cannot be deferred. However, few data concerning urgency of transfusion are available to inform planning. We designed a random sample survey to assess urgency of clinical need, and hence potential utility of triaging red cells (RC) in an emergency through restricting use only to urgent indications, for a regional population of approximately 5 million. An audit based on random sampling of units at point of distribution was used to assess state-wide RC use over time, and capture data in proportion to actual usage over both rural and metropolitan centers. Randomly selected RC units were tagged with a case-report form during production. Tagged units were distributed to hospital laboratories with routine supply. At time of issue for transfusion, the institutional laboratory completed and returned case report forms from the tagged units. Information regarding recipient demographics, indication for transfusion, urgency of supply, and (where issued to support surgery) urgency of surgery were obtained. Following an initial pilot study to refine the model, approximately 1000 tagged RC units (46% group O, 36% A, 13% B, and 4% AB; RhD positive 85% / negative 15%) of a planned 5000 total have been introduced to the state inventory. Three percent of units were not transfused. Major clinical categories of use included hematology (17% of overall use), gastroenterology (11%), orthopedic surgery (9%), non-hematological oncology (6%), gastrointestinal surgery (6%), cardiothoracic surgery (5%), obstetrics/gynecology (5%), urology (3%), trauma (3%) and pediatrics (2%); miscellaneous indications and anemia not otherwise specified constituted 10% and 22% of transfusions respectively. 18% of units were used in acute transfusion episodes (needed within 1h), 43% for urgent indications (1–24h), 33% semi-urgent (24h - 1 week) and 4% non-urgent. Of the 34% units allocated to support perioperative bleeding; only a quarter of procedures were elective. In 15% of cases the urgency of surgery was not stated. Thus, reporting institutions indicated that only 38% transfused units could have been deferred for more than 24h, and only 9% of total RCs were allocated to support potentially deferrable elective surgery. These early results suggest that triaging of RC supply in an acute shortage would only have a very short term impact on actual RC use, as the majority of RC use is already relatively urgent. Additional strategies for emergency blood contingency planning are required to meet priority clinical needs.

Description: Childhood acute myeloid leukemia (AML) has a poor prognosis with standard chemotherapy. Allogeneic bone marrow transplantation (BMT) in remission improves the outlook only for the one third of patients with sibling donors. Autologous BMT with a lower morbidity and mortality is available to all. In this study, maximum cytoreduction was achieved by intensive early chemotherapy. Final intensification, with autologous BMT was offered to all those remaining in first complete remission (CR). Patients received two induction and two consolidation courses of intensively scheduled chemotherapy. Cytoreduction was assessed on day 14 and remission was assessed after courses 2 and 4. Bone marrow was harvested after recovery from the second consolidation course or after the first maintenance course and separated on a discontinuous percoll gradient before cryopreservation. Twenty-eight of 31 consecutively enrolled patients achieved CR. Three relapsed early and, of the 25 eligible, 24 underwent autologous BMT. Twenty-three patients received high-dose melphalan and 1 received busulphan and cyclophosphamide before autologous BMT at a median of 113 days (range, 86 to 301) after initial CR. Trilineage engraftment occurred in all. Neutrophil recovery to greater than 0.5 x 10(9)/L occurred at a median of 46 days (range, 13 to 92) after autologous BMT. Platelet recovery was delayed, with a median time to achieve greater than 20 x 10(9)/L of 42 days (range, 18 to 215). With a minimum follow up of 25 months following autologous BMT only 3 children have relapsed. The 5-year event-free survival rate (EFS) from diagnosis is 68% (95% confidence interval, 46% to 90%). Five- year EFS following autologous BMT is 87% (95% confidence interval, 67% to 100%). Autologous BMT with high-dose melphalan administration after intensive chemotherapy has produced EFS equivalent to allogeneic BMT and is associated with a strikingly low relapse rate. High-dose melphalan appears to be a valuable agent for conditioning therapy in AML.

Description: In feto-maternal alloimmune thrombocytopenia (FMAIT), severe hemorrhage, particularly intracranial haemorrhage (ICH), may occur before delivery. Management strategies to prevent ICH in high-risk pregnancies include maternal administration of intravenous Ig with or without steroids and fetal platelet transfusions. This report describes a patient who lost three fetuses with ICH because of FMAIT due to anti- HPA-1a. ICH occurred earlier in successive pregnancies (at 28, 19, and 16 weeks of gestation) despite maternal treatment with intravenous Ig and steroids from 14 weeks of gestation in the third pregnancy. The fourth pregnancy was managed by administering weekly intraperitoneal injections of Ig to the fetus from 12 to 18 weeks of gestation. At 18 weeks, there was no evidence of ICH, but the fetal platelet count was only 12 x 10(9)/L. Serial fetal platelet transfusions were started, but there were poor responses because of immune destruction of the transfused platelets by maternal HLA antibodies. There were improved responses to transfusions prepared from the mother and from HLA- compatible HPA-1a-negative donors. At 35 weeks of gestation, a normal infant was delivered by Caesarean section after 20 platelet transfusions. There was prolonged thrombocytopenia in the baby for 15 weeks after birth, probably due to transfer of HPA-1a antibodies in the transfusions of unwashed maternal platelets. The optimal management of pregnancies likely to be severely affected by FMAIT is still evolving. Intensive management was successful in this case, but a successful outcome cannot be guaranteed in severely affected cases. This is the first time that HLA incompatibility has been found to complicate fetal transfusion therapy.

Description: A single umbilical cord blood unit (CBU) often doses patients at the threshhold for successful hematopoietic cell transplantation. While both cryopreserved and infused total nucleated cell (TNC) content is somewhat predictive of outcomes, there has been no reliable "potency" assay developed for UCB. Frequently, pre-cryopreserved and post-thaw recoveries are inconsistent and unpredictable. The lack of standardization from cord blood bank to bank and transplant center laboratory to laboratory further complicates interpretation of the data that is currently available. Our program is in the unique position to operate both a public cord blood bank, "The Carolinas Cord Blood Bank" (CCBB) and adult and pediatric blood and marrow transplant programs performing 〉100 unrelated donor cord blood transplants (UCBT) per year, placing us in the unique position of evaluating cord blood pre-cryopreservation and post-thaw TNC, viability, CD34 and colony forming assays (CFU-GM, CFU-GEMM, BFU-E) in the same laboratory, eliminating the potential for differences in laboratory practices. We have evaluated the results of 218 transplants performed from CBUs obtained from the CCBB and transplanted at our center. These units did not require transfer from the bank to the transplant center (TC) eliminating the possibility that warming of CBUs during shipping could compromise cell potency. In this dataset, the cummulative incidence (CINC) of neutrophil engraftment (ANC 500/uL by day +42) was 79.4% (95% CI 74–84.8) and platelet engraftment (PLT 50K by day 180) was 61.3% (95% CI 54.8–67.8). The CINC of event-free survival (EFS) at day 180 and 1 year was 68.7% (95% CI 62.5–75) and 62.5% (95% CI 56–69.1), respectively. The CI of acute grades II–IV graft-versus-host disease at days 100 and 150 was 10.6% (95% CI 6.5–14.7%). The CI of relapse at 1 year was 8.1% (95% CI 4.4–11.8%). Thus, most deaths occurred from graft failure, infection or regimen related toxicity all of which could be influenced by graft potency. We also expanded our dataset to include an additional 405 transplants where the CBU was shipped from other cord blood banks and transplanted at Duke. In this group, the CINC of neutrophil engraftment was 78.2% (95% CI 75–81.2) and the CI of EFS at day 180 and 1 year was 60–2 (95% CI 56.3–64) and 51% (95% CI 47–55), respectively. We hypothesized that post-thaw CFU and or CD34 would be better predictors of engraftment and survival than TNC. We examined the impact of post-thaw total CFU and CD34 recoveries and dosing on neutrophil engraftment and overall survival in these patients. Dosing of post-thaw CFU/kg infused (median 3.5x10e4/kg) correlated with neutrophil engraftment (p=

Description: Microparticles (MPs) are small vesicles shed from stimulated cells that permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin and differs when produced by stimulation versus apoptosis. Whether or not MP properties vary according to stimulus is not currently known. Monocyte-derived MPs are of particular interest as they are known to promote inflammation and are highly procoagulant. We studied the characteristics of MPs produced from monocytic THP-1 cells upon four different types of stimulation: lipopolysaccharide, a soluble P-selectin chimera (P-sel-Ig), an IgG control, and PBS (to study spontaneously-generated MPs). We developed a novel criterion for defining an MP using calcein-AM staining which only fluoresces when inside an intact cell or vesicle. Using proteomics, flow cytometry, western blotting, and electron microscopy, we were able to compare the properties of the four MP populations. Through our proteomic analysis, we identified 331 proteins in the MPs produced by P-sel-Ig stimulation, 830 in the MPs produced by LPS stimulation, 199 in the MPs produced by IgG stimulation, and 457 in the MPs produced spontaneously. We found that MP populations were similar with respect to size distribution and expression of certain antigens such as the β2 and αL integrins. Not only did all the MPs express cytoskeletal proteins, verified by both proteomics and western blotting, but electron microscopy revealed that these MPs contain an internal three-dimensional protein scaffolding that is similar in structure to the highly branched cytoskeleton of cells. The MPs also shared the same level of tissue factor expression and procoagulant activity. Additionally, we found that MPs have distinct characteristics depending on stimuli. The MPs differed in phosphatidylserine expression with less phosphatidylserine-positive MPs produced by P-sel-Ig stimulation. There were 52 proteins identified by proteomics to be in only the MPs produced upon P-sel-Ig stimulation, and 408 proteins found only in the MPs produced upon LPS stimulation. The MPs also differed in the expression of proteins from specific subcellular locations. For example, the MPs produced via LPS stimulation contained many more mitochondrial and nuclear proteins; whereas, the MPs produced by P-sel-Ig stimulation contained more plasma membrane proteins involved in cell adhesion and signaling. Specifically, these MPs produced by P-sel-Ig stimulation expressed the inhibitory receptor leukocyte-associated immunoglobin-like-receptor-1 (LAIR-1) which binds to collagen. The expression of LAIR-1 suggests MPs generated from P-sel-Ig stimulation could accumulate at sites of vascular injury where collagen is exposed, allowing the procoagulant MPs to promote hemostasis. Our finding that the properties of MPs depend on the stimulus that produced them supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Description: Non–Fc-receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunologic tolerance. Because factor VIII (FVIII) inhibitor formation is the most serious treatment-related complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A BALB/c and C57BL/6 mice. A short course of low-dose anti-CD3 significantly increased expression of CD25 and the proportion of CD4+CD25+ regulatory T cells in the spleen and potently prevented the production of inhibitory and non-neutralizing anti-FVIII antibodies in both strains of mouse. Depleting the CD4+CD25+ cells during anti-CD3 therapy completely ablated tolerance to FVIII. Further phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4+GITR+ and CD4+FoxP3+ cells in both strains of mice. In addition, in tolerant C57BL/6 mice we observed an increase in CD4+CD25+CTLA-4+ and CD4+CD25+mTGF-β1+ cells. Finally, in vitro cytokine profiling demonstrated that splenocytes from tolerant BALB/c and C57BL/6 were polarized toward a Th1-immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII and that the mechanism(s) regulating this response almost certainly occurs through the generation of several distinct regulatory T-cell lineages and by influencing cytokine production and profile.

Description: Abstract 2222 Activated Protein C (APC) is well known for its anticoagulant activity in the coagulation cascade. Inactivation of factors VIIIa (FVIIIa) and Va (FVa) by APC down regulates thrombin generation. The importance of FVa inactivation is seen in individuals with a common genetic mutation in Factor V, known as APC resistant Factor V Leiden (FVL). This missense mutation leads to the elimination of one of three APC cleavage sites on FVa, and FVa inactivation occurs at a slower rate. APC resistance leads to a thrombophilic state and individuals with FVL have a higher risk of thrombosis. Some reports suggest that hemophiliacs with the FVL mutation have reduced clinical severity compared to hemophiliacs without the FVL mutation. They have fewer bleeding episodes and also a delay in age when bleeding episodes begin (Kurnik et al Haematologica 2007;92:982-985). Consistent with this observation, transgenic mice engineered to carry the FVL mutation in combination with a deficiency in FVIII or factor IX (FIX) display normal thrombus formation in a laser injury model compare to no thrombus in the absence of the FVL mutation (Schlachterman et al J Thrombos and Haemostas 3:2730, 2005). Therefore, targeting a therapeutic agent to stabilize FVa by inhibiting APC may help normalize clotting in hemophiliacs. An anti-APC aptamer was discovered by Systematic Evolution of Ligands by EXponential enrichment (SELEX) and optimized for therapeutic use. In vitro assays were used to determine if the anti-APC aptamer blocks the anticoagulant activity of APC. The aptamer inhibited APC cleavage of a chromogenic peptide substrate. Furthermore, the aptamer decreased the clot time in a plasma-based assay and corrected thrombin generation as measured by calibrated automated thrombogram (CAT) following thrombomodulin-mediated protein C activation. These results show that the anti-APC aptamer efficiently blocks the anticoagulant activity of APC. APC also has an important cytoprotective role which can occur when APC binds and interacts with endothelial protein cell receptor (EPCR) and protease activated receptor-1 (PAR-1). In its cytoprotective role, APC can protect cells from apoptosis and inflammation. It is essential for the safety of a procoagulant, anti-APC therapeutic, that it not block this activity. Flow cytometric experiments were used to determine if the anti-APC aptamer affected the cytoprotective activity of APC. Aptamer was either pre-mixed with APC or added to APC-treated HUVEC cells. There was no interference of APC binding to EPCR on HUVEC cells in either type of experiment. In addition, the aptamer did not interfere with APC binding to EPCR on THP-1 cells. These experiments suggest that the anti-APC aptamer should not interfere with the ability of APC to bind to and protect cells. Taken together with the procoagulant activity described above, these data suggest that the anti-APC aptamer should be a promising new agent for the treatment of hemostatic defects. Disclosures: Wagner: Archemix Corportation: Employment. Schwartz: Archemix Corporation: Employment. McGinness: Archemix Corportation: Employment. Genga: Archemix Corporation: Employment. Kurz: Archemix Corporation: Employment. Waters: Archemix Corporation: Employment. Schaub: Archemix Corporation: Employment.

Description: Abstract 3390 Poster Board III-278 Introduction: While the outcome of children with acute lymphoblastic leukemia (ALL) has steadily improved, the prognosis for those who relapse (rALL) remains poor. Partly this has been due to the lack of a standardised approach to children with rALL. The international collaborative trial ALLR3 stratified patients into standard (SR), intermediate (IR) and high risk (HR) groups based on the time from first diagnosis to relapse, site of relapse and immunophenotype. All patients received 3 blocks of therapy, with a minimal residual disease (MRD) assessment at the end of blocks 1 and 3. Allogeneic stem cell transplantation (allo-SCT) was offered to all HR and those IR who had a MRD of ≥10-4 at week 5. In children in whom MRD was unavailable, allo-SCT was offered to those who relapsed within 24 months of stopping therapy. All other patients continued on chemotherapy for 24 months. A day 1 and 2 randomisation was performed between mitoxantrone and idarubicin. Results: 239 patients were enlisted, of whom 216 were randomised and 109 idarubicin and 103 mitoxantrone patients are analysable. There were non-significant differences between the groups with regards to time to relapse, site of relapse and cytogenetic subtypes.108 (51%) were in CR2 at a median follow up of 36 months (range 1-70) and Progression Free Survival (PFS) at 3 years was 50.3% (95% CI 42.9%, 57.3%). Forty four percent of idarubicin and 40% of mitoxantrone patients had disease levels of ≥10-4 at week 5 (p=0.90) at the end of block 1. The PFS at 3 years for those who received idarubicin was 35.9% (25.9%, 45.9%) and mitoxantrone 64.6% (54.2%, 73.2%) (p=0.0004). Adjusted for differences in risk group, country, age, gender and cytogenetic subtype, this difference continues to be significant (p=0.003). Overall mitoxantrone was better tolerated and less toxic than idarubicin. A competing risks model showed that the difference between the two drugs is primarily related to disease control (p=0.02), rather than toxicity of treatment (p=0.09). In the patients who were transplanted, 35% and 5% of patients who received idarubicin (n = 48) or mitoxantrone (n = 44) respectively have relapsed. Based on the intention to treat analyses, there were non significant differences in patients treated without an allo-SCT in both arms. IR patients who were transplanted did worse (p = 0.01) in the idarubicin group but had comparable results in the mitoxantrone group to those who received chemotherapy. As a result the randomisation in ALLR3 has now been closed, though the study continues to accrue to better answer the MRD stratification question. Conclusions: Mitoxantrone is a well tolerated and highly effective drug for the treatment of children with rALL. While MRD at week 5 failed to predict the differences in outcome for the two randomised groups, it identified that children in the IR group with