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: 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: Background: Unrelated donor umbilical cord blood is an acceptable graft source for patients lacking related donors. However, a non-engraftment rate of approximately 20% despite adequate total nucleated cell (TNC) dose remains a barrier to the overall success of UCBT. Of various patient and graft characteristics that may influence engraftment, identifying an assay predictive of cord blood unit (CBU) potency and overall engraftment would be beneficial. Methods: Pre-cryopreservation (pre-cryo) and post-thaw graft characteristics were available on 423 UCBT performed at our institution between 2/11/2000 and 5/1/2007. The units were obtained from 16 US public cord blood banks and were selected by pre-cryo cell dose and HLA matching. Pre-cryo data (TNC, CD34 cells and CFU) was provided by the cord blood bank as part of routine banking practices. All units were thawed in the Duke Stem Cell Laboratory (SCL). Post-thaw testing (TNC, CD34, CFU) was performed by consistent personnel in the SCL after thaw and washing with Dextran/Albumin as described previously by Rubinstein et al. Univariate and multivariate analyses were performed to identify significant pre-cryo, post-thaw, and baseline factors predictive of neutrophil and platelet engraftment. Results: Of the 423 evaluable patients, 68% had malignancies, 61% were males, 73% were Caucasian and 38% were CMV+. The grafts were HLA (93%), sex (50%) or racially (24%) mismatched with the patients. There was excellent correlation between pre-cryo and post-thaw TNC (r2=0.92) and CD34 (r2=0.68) content, but much weaker correlation for CFUs (r2=0.27). In univariate analysis, age (≤5 years), disease (non-malignant), weight (≤12 kg), CMV status (negative), recipient ethnicity (Caucasian), HLA match (5/6 or 6/6) and pre-cryo/post-thaw TNC (larger), pre-cryo/post-thaw CD34 (larger) and pre-cryo/post-thaw CFU (larger) were predictive of both neutrophil and platelet engraftment. Multivariate analysis of parameters are presented in Table 1. In the overall multivariate analysis of neutrophil engraftment, Male units (p=0.01), 5/6 or 6/6 HLA match (p=0.02), larger post-thaw CD34 (p=0.02) and larger post-thaw CFU (

Description: Background: Cerebral palsy (CP) results from in utero or perinatal injury to the developing brain, often through stroke, hypoxic insult, or hemorrhage. Current treatments are supportive, focusing on managing sequelae with physical therapies, medications, and surgery. However, there are no therapies to address the underlying brain injury. Umbilical cord blood (CB) has been shown to prevent neurologic damage in children with leukodystrophiesand to improve motor function in animal models of brain injury and CP. Previously, we demonstrated safety of intravenous autologous CB infusion in young children with brain injuries. The Cerebral Palsy-Autologous Cord Blood (CP-AC) study was designed to assess the efficacy of a single intravenous infusion of autologous CB in young children with spastic CP. Methods: The CP-AC study is a prospective, randomized, double blind, placebo controlled crossover study of a single intravenous infusion of autologous CB in children ages 1-6 years with spastic CP. The Gross Motor Classification System (GMFCS) was utilized to classify the level of motor function at study entry and follow-up. Children were eligible if they were (1) GMFCS level 2-4 or (2) GMFCS level 1 with hemiplegia if they used their affected hand as an assist only. Children with known genetic conditions, intractable seizures, or severe microcephaly were ineligible. Autologous CB units had to have a documented pre-cyropreservation total cell dose of ≥1x107/kg, negative sterility culture, negative maternal infectious disease screening, and confirmed identity through HLA typing of the subject and a segment attached to the CB unit. Subjects were evaluated at baseline, one year, and two years with functional evaluations (Gross Motor Function Measure-66 (GMGM-66), Peabody Developmental Motor Scales-2, Assisting Hand Assessment, Bayley Scales of Infant Development) and brain MRI. They were randomized to the order in which they received CB and placebo infusions (given one year apart). The primary endpoint was change in GMFM-66 score at one year after the initial infusion (CB or placebo). Cryopreserved CB units were shipped to and stored at Duke until the day of treatment when they were thawed and washed in dextran 40 +5% human serum albumin (DA). Infusions, dosed at 1-5x107/kg based on the pre-cryopreservation total nucleated cell count (TNCC) and diluted in 1.25mL/kg of DA, were administered intravenously over 5-10 minutes in the outpatient setting after premedication with oral acetaminophen, IV Benadryl, and IV Solumedrol. Subjects received IV fluids and were monitored for 2-4 hours post-infusion. Results: Sixty-three children were enrolled with a median age of 2 years (range 1- 6) at baseline. Median TNCC of CB infusion was 2x107/kg (range 0.4-5) with a median CD34 dose of 0.5x105/kg (range 0.05-4). Despite negative pre-cryopreservation cultures, one CB unit grew β-hemolytic strepfrom a sample of the thawed CB unit. There were no clinical infections in this or any other study patient. One subject had transient infusion reactions consisting of hives +/- low-grade fever after each infusion; an additional dose of Benadryl was administered after the first reaction. Preliminary analysis of the 63 patients at one year showed no statistically significant overall difference in GMFM-66 change scores between placebo and treated groups (6.9 vs. 7.5, p =0.72). However, treated subjects who received pre-cryopreservation cell doses of 〉2.5x107/kg demonstrated statistically significant improvement in GMFM-66 change scores compared to subjects who received lower cell doses (p 2.5x107/kg) that correspond with the minimum cell dose used for hematopoietic reconstitution in patients undergoing allogeneic CB transplantation. In order to extend this therapy to children who do not have adequate autologous CB units available, safety and efficacy of allogeneic CB products should be investigated. Figure 1. GMFM-66 Change Scores by Infused Cell Dose Figure 1. GMFM-66 Change Scores by Infused Cell Dose Disclosures No relevant conflicts of interest to declare.