Ashley Morris

Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment

BACKGROUND Delayed hematopoietic recovery is a major drawback of umbilical cord blood (UCB) transplantation. Transplantation of ex vivo-expanded UCB shortens time to hematopoietic recovery, but long-term, robust engraftment by the expanded unit has yet to be demonstrated. We tested the hypothesis that a UCB-derived cell product consisting of stem cells expanded for 21 days in the presence of nicotinamide and a noncultured T cell fraction (NiCord) can accelerate hematopoietic recovery and provide long-term engraftment. METHODS In a phase I trial, 11 adults with hematologic malignancies received myeloablative bone marrow conditioning followed by transplantation with NiCord and a second unmanipulated UCB unit. Safety, hematopoietic recovery, and donor engraftment were assessed and compared with historical controls. RESULTS No adverse events were attributable to the infusion of NiCord. Complete or partial neutrophil and T cell engraftment derived from NiCord was observed in 8 patients, and NiCord engraftment remained stable in all patients, with a median follow-up of 21 months. Two patients achieved long-term engraftment with the unmanipulated unit. Patients transplanted with NiCord achieved earlier median neutrophil recovery (13 vs. 25 days, P < 0.001) compared with that seen in historical controls. The 1-year overall and progression-free survival rates were 82% and 73%, respectively. CONCLUSION UCB-derived hematopoietic stem and progenitor cells expanded in the presence of nicotinamide and transplanted with a T cell-containing fraction contain both short-term and long-term repopulating cells. The results justify further study of NiCord transplantation as a single UCB graft. If long-term safety is confirmed, NiCord has the potential to broaden accessibility and reduce the toxicity of UCB transplantation. TRIAL REGISTRATION Clinicaltrials.gov NCT01221857. FUNDING Gamida Cell Ltd.

Natural Killer Cell-Enriched Donor Lymphocyte Infusions from A 3-6/6 HLA Matched Family Member following Nonmyeloablative Allogeneic Stem Cell Transplantation

Infusing natural killer (NK) cells following transplantation may allow less infections and relapse with little risk of acute graft-versus-host disease (aGVHD). We delivered 51 total NK cell-enriched donor lymphocyte infusions (DLIs) to 30 patients following a 3-6/6 HLA matched T cell-depleted nonmyeloablative allogeneic transplant. The primary endpoint of this study was feasibility and safety. Eight weeks following transplantation, donor NK cell-enriched DLIs were processed using a CD56(+) selecting column with up to 3 fresh infusions allowed. Toxicity, relapse, and survival were monitored. T cell phenotype, NK cell functional recovery, and KIR typing were assessed for association with outcomes. Fourteen matched and 16 mismatched transplanted patients received a total of 51 NK cell-enriched DLIs. Selection resulted in 96% (standard deviation [SD] 8%) purity and 83% (SD 21%) yield in the matched setting and 97% (SD 3%) purity and 77% (SD 24%) yield in the mismatched setting. The median number of CD3(-) CD56(+) NK cells infused was 10.6 (SD 7.91) x 10(6) cells/kg and 9.21 (SD 5.6) x 10(6) cells/kg, respectively. The median number of contaminating CD3(+)CD56(-) T cells infused was .53 (1.1) x 10(6) and .27 (.78) x 10(6) in the matched and mismatched setting, respectively. Only 1 patient each in the matched (n = 14) or mismatched (n = 16) setting experienced severe aGVHD with little other toxicity attributable to the infusions. Long-term responders with multiple NK cell-enriched infusions and improved T cell phenotypic recovery had improved duration of responses (p = .0045) and overall survival (OS) (P = .0058). A 1-step, high-yield process is feasible, and results in high doses of NK cells infused with little toxicity. NK cell-enriched DLIs result in improved immune recovery and outcomes for some. Future studies must assess whether the improved outcomes are the direct result of the high doses and improved NK cell function or other aspects of immune recovery.

Myeloablative Intravenous Busulfan/Fludarabine Conditioning Does Not Facilitate Reliable Engraftment of Dual Umbilical Cord Blood Grafts in Adult Recipients

The efficacy of once-daily intravenous busulfan with fludarabine as a preparative regimen for partially matched umbilical cord blood transplantation has not been formally studied. We randomized 10 adult patients with myeloid malignancies to receive either concurrent or sequential administration of intravenous busulfan 130 mg/m(2) once daily x 4 days and fludarabine 40 mg/m(2) daily x 4 days, followed by dual umbilical cord blood transplantation. The median combined cryopreserved total nucleated cell dose was 3.6 x 10(7)/kg recipient body weight (range: 2.8-4.5 x 10(7)/kg). Graft-versus-host disease (GVHD) prophylaxis was provided by tacrolimus and mycophenolate mofetil (MMF). Donor-derived neutrophil recovery was observed in only 2 of 10 patients, resulting in premature closure of the study as per graft failure stopping rules. We conclude that the myeloablative conditioning regimen of once-daily intravenous busulfan with fludarabine provides insufficient immunosuppression to allow for engraftment of partially matched, dual umbilical cord blood grafts.

Preemptive dosing of plerixafor given to poor stem cell mobilizers on day 5 of G-CSF administration.

Plerixafor, given on day 4 of G-CSF treatment is more effective than G-CSF alone in mobilizing hematopoietic progenitor cells. We tested a strategy of preemptive plerixafor use following assessment of the peak mobilization response to 5 days of G-CSF. Patients were eligible for plerixafor if, on day 5 of G-CSF, there were <7 circulating CD34+ cells/μL or if <1.3 × 106 CD34+ cells/kg were collected on the first day of apheresis. Plerixafor (0.24 mg/kg s.c.) was given on day 5 of G-CSF followed by apheresis on day 6. This was repeated for up to two additional doses of plerixafor. The primary end point of the study was the percentage of patients who collected at least 2 × 106 CD34+ cells/kg. Twenty candidates for auto-SCT enrolled on the trial. The circulating CD34+ cell level increased a median of 3.1 fold (range 1–8 fold) after the first dose of plerixafor and a median of 1.2 fold (range 0.3–6.5 fold) after the second dose of plerixafor. In all, 15 out of 20 (75%) patients achieved the primary end point. In conclusion, the decision to administer plerixafor can be delayed until after the peak mobilization response to G-CSF has been fully assessed.

Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation: risk factors and response to treatment

High fevers and/or rashes prior to neutrophil engraftment are frequently observed after umbilical cord blood (UCB) transplantation, and the condition is referred to as pre-engraftment syndrome (PES). Few studies have evaluated the risk factors for and treatment response to PES. Therefore, we retrospectively characterized PES in 57 consecutive engrafted patients (⩾12 years old) who received myeloablative dual UCB transplantation. All patients received TBI (⩾13.2 Gy)-based myeloablative conditioning. Tacrolimus (n=35) or CYA (n=22) combined with mycophenolate mofetil was used as GVHD prophylaxis. PES was defined as the presence of non-infectious fever (⩾38.5 °C) and/or rash prior to or on the day of neutrophil engraftment. The incidence (95% confidence interval) of PES was 77% (66–88%). The incidence of PES was significantly higher in patients who received CYA as a GVHD prophylaxis than those who received tacrolimus (P<0.001), and this association was confirmed in the multivariate analysis. The occurrence of PES did not impact OS or tumor relapse, although it may have increased non-relapse mortality (P=0.071). The incidence of acute GHVD or treatment-related mortality was not influenced by the choice to use corticosteroids to treat PES. This study suggests that use of CYA for GVHD prophylaxis increases the risk of PES following dual UCB transplantation.

Granisetron vs ondansetron for prevention of nausea and vomiting in hematopoietic stem cell transplant patients: results of a prospective, double-blind, randomized trial.

Summary:The serotonin type-3 (5-HT3) antagonists represent a significant advance in the prevention of acute nausea and vomiting (N/V) from highly emetogenic chemotherapy. We sought to determine if any differences in efficacy or adverse effects exist between two such agents, ondansetron and granisetron, during conditioning therapy for hematopoietic stem cell transplantation (HSCT). Patients were randomized to receive either ondansetron 0.15 mg/kg intravenously every 8 h or granisetron 10 μg/kg intravenously daily. Additionally, all patients received scheduled dexamethasone and lorazepam. Prophylaxis was continued until 24 h after completion of chemotherapy. Nausea and distress were measured subjectively with visual analog scales and emetic episodes were quantified. Of the 110 randomized patients, 96 were evaluable for efficacy and safety. No significant differences in efficacy were observed between the ondansetron- and granisetron-treated patients, evaluated by comparing the degree of nausea and distress, number of emetic episodes and overall control of emesis. The adverse effects were also comparable and no patients were removed from study because of severe toxicities. This trial demonstrates that ondansetron and granisetron are equally effective at preventing acute N/V associated with conditioning therapy frequently used for HSCT. The agent of choice should be based on drug acquisition cost or preference.

Outcomes of a 1-day nonmyeloablative salvage regimen for patients with primary graft failure after allogeneic hematopoietic cell transplantation

Primary graft failure after allogeneic hematopoietic cell transplantation is a life-threatening complication. A shortened conditioning regimen may reduce the risk of infection and increase the chance of survival. Here, we report the outcome of 11 patients with hematologic diseases (median age, 44; range, 25–67 years, seven males) who received a 1-day reduced-intensity preparative regimen given as a re-transplantation for primary graft failure. The salvage regimen consisted of fludarabine, cyclophosphamide, alemtuzumab and TBI, all administered 1 day before re-transplantation. All patients received T-cell replete PBSCs from the same or a different haploidentical donor (n=10) or from the same matched sibling donor (n=1). Neutrophil counts promptly increased to >500/μL for 10 of the 11 patients at a median of 13 days. Of these, none developed grade III/IV acute GVHD. At present, 8 of the 11 patients are alive with a median follow-up of 11.2 months from re-transplantation and 5 of the 8 are in remission. In conclusion, this series suggests that our 1-day preparative regimen is feasible, leads to successful engraftment in a high proportion of patients, and is appropriate for patients requiring immediate re-transplantation after primary graft failure following reduced-intensity transplantation.

Mini test dose of intravenous busulfan (busulfex(®)) in allogeneic non-myeloablative stem cell transplantation, followed by liquid chromatography tandem-mass spectrometry.

Busulfan (Bu) has been used for almost 3 decades in the conditioning of hematopoietic stem cell transplant. Bu has a very diverse gastrointestinal absorption which in association with its emetic effect in high doses makes inter- and intra-individual oral bioavailability and thus systemic exposure very variable and difficult to predict (1, 2, 3). If the systemic exposure is below the therapeutic window there is an increased risk of lack of engraftment (4) and, in chronic myelogenous leukemia (CML) patients, disease relapse (5), while overexposure results in an increased incidence of high-dose related toxicities like hepatic veno-occlusive disease (6, 7). It is important to note that “busulfan concentration-response relationships are regimen-, age- and disease-dependent”, as emphasized by McCune et al (8). Therapeutic dose monitoring (TDM) gained an important role in the setting of oral Bu dose as it became clear that the level of systemic exposure influenced the toxicity, engraftment and relapse rates. To give an idea of the importance of TDM, a test dose prior to starting the conditioning regimen with home-administered oral Bu in 153 patients evidenced that as many as 68% of them would have drug exposures out of the therapeutic window (900–1,500 μM·min) with the standard dose of 1mg/kg of ideal body weight every 6 hours (9). An intravenous (i.v.) formulation was developed to overcome these issues and avoid the utilization of TDM, However, in a series of 6 studies using intravenous Bu in adults, 72 to 91% of the patients achieved the target AUC without dose changes, while 10 to 82% of children achieved it in a series of 5 studies with the intravenous drug (10). Clearly, a need for TDM even when i.v. is used still persists. Many different strategies using single or repeated sampling in different points of the therapy were proposed to improve TDM results (10). However, since TDM can only be useful if Bu determinations are timely delivered and many transplant centers do not have a facility to perform Bu determination, new approaches using pharmacokinetic analysis of a sub-therapeutic test dose administered a few days earlier to predict the ideal dose were proposed. In one such study, 7 patients received a test dose of 0.8 mg/kg, followed by 0.8 mg/kg/dose every 6 hours for 15 doses (11). Bu concentrations were obtained and mean drug clearance was determined after the test, first and 13th doses. The utilization of a test dose demonstrated bioequivalence ( 20% variability). However, when the same 0.8 mg/kg test dose assay was compared to the intravenous Bu in continuous infusion for 90 hours in other 7 patients, they showed similar clearances (12). The next step was to examine the ability of the test dose clearance to base the calculation of the 90 hour continuous infusion dose (13). All 4 patients tested had AUCs within the desired range and no adjustments were required after 18 hours. More recently, Beri et al. (14) used a 0.8 mg/kg i.v. test dose (1/4 of the standard daily dose). Without the test dose, 23% of the 17 patients would have fallen below and 12% above the therapeutic range. With the test dose, no patients had a sub-therapeutic AUC and only 12% had an AUC above the therapeutic window. In this work, in order to further reduce the unnecessary exposure and adverse effects of the test dose process, we investigated bioequivalence of a very low sub-therapeutic test dose (1/10 of the standard daily dose) of i.v. Bu (Busulfex®). For this purpose we developed a sensitive and fast liquid chromatography tandem-mass spectrometry (LC-MS/MS) method which allowed us to measure Bu in a wide concentration range required in the study.

Transplantation of Ex Vivo Expanded Umbilical Cord Blood (NiCord) Decreases Early Infection and Hospitalization

Delayed hematopoietic recovery contributes to increased infection risk following umbilical cord blood (UCB) transplantation. In a Phase 1 study, adult recipients of UCB stem cells cultured ex vivo for 3 weeks with nicotinamide (NiCord) had earlier median neutrophil recovery compared with historical controls. To evaluate the impact of faster neutrophil recovery on clinically relevant early outcomes, we reviewed infection episodes and hospitalization during the first 100 days in an enlarged cohort of 18 NiCord recipients compared with 86 standard UCB recipients at our institution. The median time to neutrophil engraftment was shorter in NiCord recipients compared with standard UCB recipients (12.5 days versus 26 days; P < .001). Compared with standard UCB recipients, NiCord recipients had a significantly reduced risk for total infection (RR, 0.69; P = .01), grade 2-3 (moderate to severe) infection (RR, 0.36; P < .001), bacterial infection (RR, 0.39; P = .003), and grade 2-3 bacterial infection (RR, 0.21; P = .003) by Poisson regression analysis; this effect persisted after adjustment for age, disease stage, and grade II-IV acute GVHD. NiCord recipients also had significantly more time out of the hospital in the first 100 days post-transplantation after adjustment for age and Karnofsky Performance Status (69.9 days versus 49.7 days; P = .005). Overall, transplantation of NiCord was associated with faster neutrophil engraftment, fewer total and bacterial infections, and shorter hospitalization in the first 100 days compared with standard UCB transplantation. In conclusion, rapid hematopoietic recovery from an ex vivo expanded UCB transplantation approach is associated with early clinical benefit.

4-Hydroperoxycyclophosphamide–purged peripheral blood stem cells for autologous transplantation in patients with acute myeloid leukemia

We have performed a phase I dose escalation of 4-Hydroperoxycyclophosphamide (4HC) purging of autologous peripheral blood progenitor cells (PBPCs) to improve the outcome of autologous transplantation for patients with myeloid leukemia. Peripheral blood stem cells were mobilized after cytosine arabinoside of 2 g/m(2) every 12 hours x 8 doses with etoposide of 40 mg/kg total dose infused over 4 days, followed by growth factor support. The preparative regimen included Busulfan of 1 mg/kg orally every 6 hours x 16 doses, followed by etoposide of 60 mg/kg x 1 day (the patient with chronic myeloid leukemia received cyclophosphamide of 60 mg/kg/d x 2 days in lieu of etoposide). PBPCs purged with 4HC were infused following this induction. Toxicities included grade 3 or 4 skin rashes, stomatitis/mucositis, and delay in time to hematopoietic recovery. The maximum tolerated dose of 4HC used to purge PBPCs in this trial was 20 microg/mL, which resulted in an average of 18 days for white blood cells and 28 days for platelet recovery. With a median follow-up of 2.25 years in surviving patients, the 3-year disease free survival rate is 44% and the overall survival rate is 89%. These data suggest that autologous PBPCs are more sensitive than marrow purged with 4HC, tolerating less intense purging, although a survival advantage may still be seen and should be assessed in larger studies. Approaches to minimize stomatitis and protect normal stem cells from the toxicity of 4HC may improve the tolerance and efficacy of this approach.