Busulfan Dose Research Articles

Histological Study on the Protective Effect of Endogenous Stem Cell Mobilization in Busulfan-Induced Testicular Injury in Albino Rats.

Background:Testicular damage is one of the most hazardous effects of chemotherapy as it is frequently associated with oligozoospermia and azoospermia.Aim of the Work:This study aimed at evaluating the protective effect of hematopoietic stem cell mobilization by granulocyte colony-stimulating factor (G-CSF) in a rat model of busulfan-induced testicular injury.Materials and Methods:Twenty-four adult albino rats were divided into four groups: group I, the control, Group II: rats received two doses of busulfan (each 15 mg/kg) intraperitoneally (IP) with 14 days interval, Group III: rats received busulfan and left untreated, and Group IV received busulfan IP then G-CSF (70 μg/kg/day) subcutaneously for 5 consecutive days. Testicular sections were stained with H and E and immunohistochemically for CD34, proliferating cell nuclear antigen (PCNA) and caspase-3, and semithin sections were stained with toluidine blue.Results:Groups II and III showed loss of the normal histological architecture of the testis and spermatogenic cells, with increased apoptosis confirmed by significantly increased caspase-3 and significantly decreased PCNA immunoexpression. While Group IV revealed improved testicular histology, decreased apoptosis, and increased proliferative capacity of spermatogenic cells. This was confirmed by significantly decreased caspase-3 immunoexpression and increased PCNA immunoreaction.Conclusion:Mobilization of stem cells with G-CSF was found to improve the testicular histology following busulfan chemotherapy in albino rats.

Busulfan Dosing Literature Review

Introduction: There is debate regarding the use of busulfan every 6 hours (q6 h) compared to every 24 hours (q24 h) in patients undergoing hematopoietic cell transplantation.Objectives: To review the literature to determine whether there is a significant difference between dosing busulfan q6 h vs. q24 h in adults, and to review dosing strategies to optimize daily dosing.Methods: A literature search was conducted in PubMed with the terms “busulfan” and “transplant” and “24” in all fields. Results were further refined by using the terms “busulfan” and “transplant” and “pharmacokinetics”. Titles were then reviewed for relevance, and the remaining articles reviewed by abstract. Articles deemed relevant were then read in more thorough detail, and references cited by these articles reviewed to ensure a comprehensive review of the literature. Studies focusing on the pediatric population were not reviewed.Results: 478 articles were identified, and of these, 372 contained the term “pharmacokinetics”. Based on abstract review, 26 relevant articles were identified. All articles confirmed that there are no differences in the pharmacokinetics of q6 h vs. q24 h dosing, and that safety appears equivalent between the two dosing schemes. One study noted an increase in the occurrence of acute graft-vs.-host disease (GVHD) and possibly increased gastrointestinal (GI) toxicity and stomatitis with q6 h dosing, while another noted a higher incidence of toxicity with q24 h dosing specifically in metastatic renal cell carcinoma patients. All articles concluded that both regimens are equally effective, but that q24 h dosing is more convenient and likely to decrease hospitalization, nursing, and pharmacy requirements.Conclusions: There is no difference in efficacy or safety between busulfan q6 h and q24 h dosing. Institutions should consider moving to daily dosing of busulfan for improved convenience and decreased costs.

The application of pharmacokinetics for Busulfan dose adjustment in hematopoietic stem cell transplantation (HSTC) in Thai children

The application of pharmacokinetics for Busulfan dose adjustment in hematopoietic stem cell transplantation (HSTC) in Thai children

Thiotepa-busulfan-fludarabine compared to busulfan-fludarabine for sibling and unrelated donor transplant in acute myeloid leukemia in first remission.

BackgroundA preparatory regimen consisting of thiotepa-busulfan-fludarabine (TBF) has been associated with reduced relapse in patients with haematological malignancies after haploidentical and cord blood transplants; however, few data exist regarding TBF conditioning in sibling (MSD) and unrelated donor (URD) transplants for AML.ResultsAmong patients receiving a myeloablative (MAC) regimen, TBF-MAC was associated with significantly lower relapse (HR 0.47, p = 0.005) however higher non-relapse mortality (NRM, HR 2.69, p < 10–4) as compared to BF. This led to similar leukemia-free (LFS) and overall survival (OS) between the two regimens (LFS: p = 0.6; OS: p = 0.27). When we selected TBF-MAC patients receiving busulfan 9.6 mg/kg, NRM resulted still higher but no more significantly different as compared to BF-MAC with busulfan 12.8 mg/kg (HR 1.53, p = 0.12); despite the lower busulfan dose, relapse remained inferior with TBF-MAC (HR 0.45, p = 0.01), however no difference in survival could be demonstrated (LFS: p = 0.31; OS: 0.82). Among patients receiving a reduced-intensity (RIC) regimen, similar outcome was observed with TBF-RIC and BF-RIC (LFS: p = 0.77; OS: p = 0.88).ConclusionsTBF-MAC as conditioning regimen for transplant from MSD and URD in AML patients in first remission provided stronger anti-leukemic activity but higher NRM as compared to BF-MAC, thus leading to similar survival. TBF-MAC with busulfan 9.6 mg/kg was associated with low relapse and acceptable NRM, however again with no survival benefit. TBF-RIC and BF-RIC resulted in comparable outcome.MethodsWe conducted a registry-based study comparing outcomes of patients with AML in first remission undergoing transplant from MSD or URD prepared with either TBF (n = 212) or BF (n = 2698) conditioning.

Test Dose Pharmacokinetics in Pediatric Patients Receiving Once-Daily IV Busulfan Conditioning for Hematopoietic Stem Cell Transplant: A Reliable Approach?

Intravenous (IV) busulfan test dose pharmacokinetics (PK) has been shown to accurately predict once-daily dose requirements and improve outcomes in adult transplant patients, but there are limited data to support this approach in children. Test doses of busulfan ∼0.8mg/kg were infused over 2 to 3hours, followed by serial sampling to 4-6hours postinfusion in pediatric hematopoietic stem cell transplant recipients (n=5). Once-daily busulfan doses were calculated based on a myelosuppressive area under the concentration-time curve (AUC) target of ∼3700 to 4000μmol·min/L and assumed dose-proportionality to the test dose. PK analysis was then repeated at full daily doses within 6-8 days of test dose administration. Plasma PK samples collected under test and full-dose conditions were analyzed using validated commercial assays and noncompartmental methods. In 4 out of 5 patients, PK estimates after once-daily IV busulfan administration differed in comparison to test dose estimates (AUC range -38.2% to +49.7%, clearance range -34.3% to +61.8%). Patients 1, 2, and 3 required increases in remaining daily busulfan doses to achieve AUC targets, and no adjustment was required in patient 4. Patient 5's AUC was 49.7% higher than expected, and he subsequently developed fatal sinusoidal obstruction syndrome. In our experience with pediatric patients, test dose PK failed to reliably predict daily dosing requirements with large discrepancies from predicted AUC targets. This article highlights the necessity for therapeutic drug monitoring of IV busulfan and inadvisability of relying solely on test-dose busulfan PK in pediatric patients. Furthermore, clinicians should consider strategies to expedite dose adjustments in real time.

200 Optimization of Individual Stages of Chicken Transgenesis to Increase Efficiency

Primordial germ cells (PGC) are the precursors of male and female progenitor cells. The cells are considered a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the PGC from chick donor embryos, transformation of the cells, and injection into the dorsal aorta of recipient embryos. After injection, the PGC are involved in the process of embryo development and differentiate into male or female sex cells. The aim of the research was to optimize the individual stages of this technology to increase the efficiency of transgenesis. The PGC were extracted from embryo gonads at stage 26 to 27 (H&amp;H) using the trypsinization process. The trypsin concentration and incubation time were determined experimentally. Treatment of chick embryos with a 0.05% trypsin solution for 5 min was optimal for obtaining culture of embryonic cells. Separation of the PGC from other types of embryonic cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population for further cultivation was established by transfer (twice) of the supernatant containing unattached cells after 1 h of cultivation in a new culture dish. The cell population is represented mainly by the PGC (81 ± 4%). Additional purification of the PGC from other cell types using magnetic-activated cell sorting (MACS) increased the proportion of these cells up to 93 ± 2%. The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the PGC. The efficiency of infection of PGC with lentiviral particles (TU/mL = 2.5 × 108) was 70 ± 3%. The transformed cells were injected into the dorsal aorta of recipient embryos on Day 2.5 (n = 80). Before injecting donor PGC, recipient embryos were treated with busulfan to remove the endogenous PGC. The optimal dose of busulfan was selected experimentally. A series of experiments introducing busulfan in concentrations from 50 to 250 μg into chick embryos at 24 h of incubation showed that the optimal dose was 100 μg/embryo. The efficacy of colonization of gonads with donor PGC was assessed on Day-10 embryos (n = 32) and 4-week-old hatched chickens (n = 12). Cells from gonads were studied using fluorescence microscopy, fluorescence-activated cell sorting (FACS) and qPCR. The presence of fluorescent cells in the gonads of recipients was established in both embryos and hatched chickens. The relative number of the recombinant DNA copies and the relative level of expression were confirmed by qPCR. The FACS analysis of sex cells isolated from gonads of recipients showed that the percentage of transformed germ cells reached 55.8% in females (n = 5) and 31.9% in males (n = 7). Thus, the effectiveness of poultry transgenesis can be enhanced by preparation of donor PGC for injection into embryo recipients and elimination of endogenous PGC in recipients. Both the purification of PGC from other cell types based on adhesive capacity as well as treatment of embryo recipients at 24 h incubation with busulfan (100 μg/embryo) increased the effectiveness of transgenesis. Study supported by the RSF within project No. 16-16-10059.

Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells.

STUDY QUESTIONIs testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice?SUMMARY ANSWERCancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls.WHAT IS KNOWN ALREADYSpermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically.STUDY DESIGN, SIZE, DURATIONThis study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months.PARTICIPANTS/MATERIALS, SETTING, METHODSNeonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497–517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR.MAIN RESULTS AND THE ROLE OF CHANCEWe found 9% (95% CI: 2–25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14–45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1–1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076).LARGE SCALE DATANA.LIMITATIONS, REASONS FOR CAUTIONAlthough we attempted to mimic the future clinical application of SSCT in humans as close as possible, the mouse model that we used might not reflect all aspects of the future clinical setting.WIDER IMPLICATIONS OF THE FINDINGSThe absence of an increase in cancer incidence and a decrease in survival of mice that received a testicular transplantation of in vitro propagated SSCs is reassuring in light of the future clinical application of SSCT in humans.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by KiKa (Kika86) and ZonMw (TAS 116003002). The authors report no financial or other conflict of interest relevant to the subject of this article.

Toward Individualizing Conditioning Regimens in Reduced-Intensity Transplantation

The advent of reduced intensity conditioning (RIC) in the 1990's has expanded accessibility to transplant thereby permitting previously ineligible patients to receive such therapy. Consensus guidelines defining dose intensity were subsequently developed (1, 2). Fludarabine combined with intermediate dose busulfan (FB) or intermediate dose melphalan (FM) are the most commonly used RIC regimens. Although these protocols are considered equivalent in terms of myelosuppressive and immunosuppressive properties, there is no prospective comparative data to further guide their use. Therefore, the choice of RIC regimen relies heavily on the preferences and experience of the transplant center.

GSTA1 diplotypes affect busulfan clearance and toxicity in children undergoing allogeneic hematopoietic stem cell transplantation: a multicenter study.

Busulfan (BU) dose adjustment following therapeutic drug monitoring contributes to better outcome of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-guided BU dose adjustments. To investigate this aspect, polymorphism within glutathione S transferase genes were assessed. Particularly, promoter haplotypes of the glutathione S transferase A1 (GSTA1) were evaluated in vitro, with reporter gene assays and clinically, in a pediatric multi-center study (N =138) through association with BU pharmacokinetics (PK) and clinical outcomes. Promoter activity significantly differed between the GSTA1 haplotypes (p<0.001) supporting their importance in capturing PK variability. Four GSTA1 diplotype groups that significantly correlated with clearance (p=0.009) were distinguished. Diplotypes underlying fast and slow metabolizing capacity showed higher and lower BU clearance (ml/min/kg), respectively. GSTA1 diplotypes with slow metabolizing capacity were associated with higher incidence of sinusoidal obstruction syndrome, acute graft versus host disease and combined treatment-related toxicity (p<0.0005). Among other GST genes investigated, GSTP1 313GG correlated with acute graft versus host disease grade 1-4 (p=0.01) and GSTM1 non-null genotype was associated with hemorrhagic cystitis (p=0.003). This study further strengthens the hypothesis that GST diplotypes/genotypes could be incorporated into already existing population pharmacokinetic models for improving first BU dose prediction and HSCT outcomes. (No Clinicaltrials.gov identifier: NCT01257854. Registered 8 December 2010, retrospectively registered).

Different approaches to establish infertile rooster

Several methods have been developed to suppress spermatogenesis in recipient males before spermatogonial stem cells (SSCs) transplantation. The aim of this study was to compare two different methods of depleting endogenous spermatogenesis in recipient ROSS 308 strain adult roosters. Gamma-radiation and alkylating agent busulfan were utilized to infertilize adult roosters (ROSS 308 strain). Two radiation therapy regimes (based on 60co isotope) were conducted locally to testes using 40Gy (5×8Gy with three-day intervals) and 30Gy (3×10Gy with three-day intervals). And two different levels of busulfan 60mg(40+20) and 50mg(30+20) with 10-day intervals were injected intraperitoneally. The results showed that both radiation therapy regimes and both busulfan levels reduced sperm motility and sperm concentration significantly compared with control group. Moreover, there were no significant differences between gamma radiation and busulfan treatments in progressive and total motility of sperm reduction. Sperm concentration reached to zero at the end of the 4th week of experiment in all treatment groups. Also histological examinations revealed that both treatments could significantly reduce the diameter of seminiferous tubules and thickness of epithelium. None of the treatments had significant effect on body weight in comparison with control group and the health status of experimental roosters remained good throughout the study. Given that, the risk probability of high doses of radiation exposure and busulfan, it can be concluded that the 30Gy (3×10Gy) and 50mg (30+20) are appropriate for suppression of endogenous spermatogenesis in mature roosters.

Depletion of endogenous germ cells in tree shrews in preparation for spermatogonial transplantation.

To achieve successful spermatogonial transplantation, endogenous germ cells must be depleted in recipient animals to allow donor germ cells to colonize efficiently. Busulfan is commonly used for the depletion of endogenous germ cells in recipient males. However, the optimal dose of busulfan is species-specific, and the optimal dose in tree shrews is yet to be determined. The current study aimed to determine the optimal dose of busulfan for effective suppression of endogenous spermatogenesis in tree shrews. Different doses (15, 20, 25, 30, 35, 40 and 45 mg/kg) of busulfan were injected into tree shrews intraperitoneally. Survival rates of the different treatment groups were calculated at 2 weeks and body weights were measured at 4, 6, 8, 10 and 28 weeks post-busulfan treatment. The testes were also removed and weighed at 4, 6, 8, 10 and 28 weeks post-treatment, and the cross and longitude diameters of the testes and diameters of the seminiferous tubules were measured and histologically evaluated. It was observed that there were no significant differences in the survival rates between the 15-35 mg/kg treatment groups and the control group (P>0.05), while the survival rate of the 40 mg/kg treatment group significantly decreased relative to the control group (P<0.05) and the survival rate of the 45 mg/kg treatment group was 0% (P<0.05 vs. control). In addition, the weight and diameters of the testes, diameters of the seminiferous tubules and proportion of normal type tubules in the 40 mg/kg group significantly decreased over 4-10 weeks relative to the control group (P<0.05), though gradually recovered with time. At 28 weeks, the recovery was significant relative to 4 weeks (P<0.05). Similarly, histological analysis indicted that recovery of abnormal tubules was delayed in the 40 mg/kg group relative to the lower dose groups as the 40 mg/kg dose tree shrews had more tubules with no spermatogenesis compared with the lower dose group at the same time points. These data indicate that a busulfan dose of 40 mg/kg is optimal for the depletion of endogenous germ cells in tree shrews. This dose led to maximum suppression of endogenous spermatogenesis while maintaining an acceptable survival rate of >50% of the lethal dose of busulfan for tree shrews.

Impact of immunoablation and autologous hematopoietic stem cell transplantation on gray and white matter atrophy in multiple sclerosis

Background: Immunoablation and autologous hematopoietic stem cell transplantation (IA/aHSCT) halts relapses, white matter (WM) lesion formation, and pathological whole-brain (WB) atrophy in multiple sclerosis (MS) patients. Whether the latter was due to effects on gray matter (GM) or WM warranted further exploration. Objective: To model GM and WM volume changes after IA/aHSCT to further understand the effects seen on WB atrophy. Methods: GM and WM volume changes were calculated from serial baseline and follow-up magnetic resonance imaging (MRI) ranging from 1.5 to 10.5 years in 19 MS patients treated with IA/aHSCT. A mixed-effects model with two predictors (total busulfan dose and baseline T1-weighted WM lesion volume “T1LV”) characterized the time-courses after IA/aHSCT. Results: Accelerated short-term atrophy of 2.1% and 3.2% occurred in GM and WM, respectively, on average. Both busulfan dose and T1LV were significant predictors of WM atrophy, whereas only busulfan was a significant predictor of GM atrophy. Compared to baseline, a significant reduction in GM atrophy, not WM atrophy, was found. The average rates of long-term GM and WM atrophy were −0.18%/year (standard error (SE): 0.083) and −0.07%/year (SE: 0.14), respectively. Conclusion: Chemotherapy-related toxicity affected both GM and WM. WM was further affected by focal T1-weighted lesion-related pathologies. Long-term rates of GM and WM atrophy were comparable to those of normal-aging.

Pediatric patients undergoing hematopoietic stem cell transplantation can greatly benefit from a novel once-daily intravenous busulfan dosing nomogram.

Busulfan, a bifunctional alkylating agent, has been used as a conditioning regimen prior to allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to derive a novel once-daily intravenous (IV) busulfan dosing nomogram for pediatric patients undergoing HSCT using a population pharmacokinetic (PK) model. A population PK analysis was performed using 2183 busulfan concentrations in 137 pediatric patients (age: 0.6-22.2 years), who received IV busulfan once-daily for 4 days before undergoing HSCT. Based on the final population PK model, an optimal once-daily IV busulfan dosing nomogram was derived. The percentage of simulated patients achieving the daily target area under the concentration-time curve (AUC) by the new nomogram was compared with that by other busulfan dosing regimens including the FDA regimen, the EMA regimen, and the empirical once-daily regimen without therapeutic drug monitoring (TDM). A one-compartment open linear PK model incorporating patient's body surface area, age, dosing day, and aspartate aminotransferase as a significant covariate adequately described the concentration-time profiles of busulfan. An optimal dosing nomogram based on the PK model performed significantly better than the other dosing regimens, resulting in >60% of patients achieving the target AUC while the percentage of patients exceeding the toxic AUC level was kept <25% during the entire treatment period. A novel once-daily busulfan dosing nomogram for pediatric patients undergoing HSCT is useful for clinicians, particularly in a setting where TDM service is not readily available or to optimize the dose on day 1.

Limited sampling strategy for predicting busulfan exposure in hematopoietic stem cell transplantation recipients.

Background Optimization of the area under the concentration-time curve (AUC) of busulfan, an essential component of conditioning regimens, improves the outcomes in patients undergoing hematopoietic stem cell transplantation (HSCT). Traditional sampling methods for calculating AUC require multiple sampling. Objective To establish a limited sampling strategy for predicting the AUC0-12 of intravenous busulfan for Chinese adult patients prior to HSCT. Methods The pharmacokinetics of twice-daily intravenous busulfan was studied in forty-five Chinese adult patients. Limited sampling models were established by the multiple linear regression analysis. The prediction error (PE) and the absolute prediction error (APE) were calculated to evaluate predictive accuracy. The agreement between the predicted and actual AUC0-12 was investigated by the Bland-Altman analysis. The accuracy and robustness of the models was validated by the bootstrap analysis. Results The AUC0-12 values of the 1st and 7th doses of busulfan were 1491±403.7 and 1908±518.5μmolL-1min, respectively. The 2-sample model suggested that the predicted AUC0-12 of twice-daily intravenous busulfan could be calculated using the following equation: AUC0-12=40.017+0.955×C3+1.088×C6 with r2=0.919. The mean PE and APE of the model were 0.52±7.67 and 6.32±4.27%, respectively. Conclusion The 2-sample model is an effective and reliable approach to predict the AUC0-12 of twice-daily intravenous busulfan in Chinese adult patients.

A Minimum Conditioning Protocol towards Transplantation Tolerance in NOD Mice by Mixed Hematopoietic Chimerism

Abstract Stable mixed hematopoietic chimerism is a robust method for inducing donor specific tolerance. However, its clinical application is dampened by the toxicity of current recipient conditioning regimens. We previously showed an irradiation-free mixed chimerism protocol in diabetes prone NOD mice is achievable with antibodies to T cells and CD40L together with busulfan and high dose rapamycin. We sought to generate a more clinically feasible chimerism protocol and tested the hypothesis that more efficient recipient T cell depletion would eliminate the need for anti-CD40L (known to cause thromboembolism in humans) and rapamycin. We preconditioned NOD mice with donor specific transfusion from fully mismatched mice (d −10), cyclophosphamide (CYP) (d −8), antibodies against CD90 and/or CD4 + CD8 (d −6, −1, 4, 9, 14), busulfan (d −1) and donor bone marrow transplant (d 0). Flow cytometry was used to detect chimerism. Mixed chimerism was induced in 33/45 NOD mice. Stable chimerism with multilineage donor cells was maintained in 21/33 recipients. Loss of chimerism could be predicted by a lower early level of chimerism at d 4, 9 or 14. Inclusion of αCD90 mAb, busulfan and CYP was critical for chimerism induction. With anti-CD90 17/23 mice became stable chimeras. 5/5 chimeric mice accepted skin from bone marrow donors but rejected skin from MHC-matched and minor antigen mismatched donors. The loss of anti-donor Vβ11+ T cells and anti-recipient Vβ17+ T cells in stable chimeric mice indicated the establishment of chimerism involves clonal deletion. Conclusion A protocol causing rapid and robust recipient T cell depletion generated chimerism without the need for anti-CD40L or rapamycin in tolerance induction resistant NOD mice.

Radiation-free, alternative-donor HCT for Fanconi anemia patients: results from a prospective multi-institutional study

AbstractFanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by chromosomal fragility, progressive marrow failure, and cancer predisposition. Hematopoietic cell transplantation (HCT) is curative for FA-related marrow failure or leukemia, but both radiation exposure during transplant and graft-versus-host disease (GVHD) may increase risk of later malignancies of the head and neck and anogenital area. In this study, we tested a radiation-free conditioning regimen with a T-cell–depleted graft to eliminate radiation exposure and minimize early and late toxicities of transplant. Forty-five patients (median age, 8.2 years; range 4.3-44) with FA underwent HCT between June 2009 and May 2014. The preparative regimen included busulfan, cyclophosphamide, fludarabine, and rabbit anti-thymocyte globulin. Busulfan levels were monitored to avoid excess toxicity. All grafts were CD34-selected/T-cell–depleted using the CliniMacs CD34 columns (Miltenyi). Thirty-four patients (75.6%) with marrow failure and 11 (24.4%) with myelodysplastic syndrome underwent HCT using matched unrelated (n = 25, 55.5%), mismatched unrelated (n = 14, 31.1%), or mismatched related donors (n = 6, 13.4%). One year probabilities of overall and disease-free survival for the entire cohort, including patients with myeloid malignancy and those receiving mismatched related/haploidentical grafts, were 80% (±6%) and 77.7% (±6.2%), respectively (median follow-up 41 months). All young children (<10 years of age) undergoing HCT for marrow failure using low-dose busulfan-containing regimen survived. No patients developed acute grade 3-4 GVHD. Sequential reduction of busulfan dose was successfully achieved per study design. Our results show excellent outcomes in patients with FA undergoing alternative donor HCT without radiation exposure. The study is registered to www.clinicaltrials.gov as #NCT01082133.

Therapeutic drug monitoring-guided dosing of busulfan differs from weight-based dosing in hematopoietic stem cell transplant patients

Busulfan (Bu)-based preparative regimens in hematopoietic stem cell transplantation are commonly used. Previous studies have shown that Bu at a fixed dose of 3.2mg/kg/day (FBD) given intravenously decreases variability in drug pharmacokinetics and this decreases the dependency on therapeutic drug monitoring (TDM) of Bu. We compared the Bu dose given using TDM with the FBD of 3.2mg/kg/day. Seventy-three patients with acute leukemia, myelodysplasia, chronic myeloid leukemia, thalassemia major, and sickle cell disease were included. The mean age at transplant was 15years (range 2–55years) with 57% adults. Indication for transplantation was leukemia/myelodysplastic syndrome in 46% of the patients, while the remaining 54% were transplanted for inherited blood disorders. We found that the median FBD was lower than the median TDM dose by 39mg/day with a statistically significant difference (p<0.001) even after adjusting for the weight (median total FBD of 349mg, median TDM dose of 494mg, p<0.0001). Age and underlying condition (malignant vs. nonmalignant) were the main factors affecting Bu clearance (p<0.001 and p<0.07, respectively). TDM remains an important tool for the appropriate dosing of Bu in preparative regimens of hematopoietic stem cell transplantation, especially in populations with genetic admixture.

Bone Marrow-Derived Cell Accumulation in the Spinal Cord Is Independent of Peripheral Mobilization in a Mouse Model of Amyotrophic Lateral Sclerosis

Bone marrow-derived cells (BMDCs) are capable of migrating across the blood–brain barrier (BBB) and accumulating in the central nervous system (CNS) when transplanted into recipients conditioned with whole-body irradiation or chemotherapy. We used the chemotherapeutic agents busulfan and treosulfan to condition recipient mice for transplantation with bone marrow (BM) cells isolated from donor mice ubiquitously expressing green fluorescent protein. We attempted to increase the accumulation of BMDCs in the CNS by mobilization of BMDCs using either, or both, granulocyte colony-stimulating factor (GCSF) or plerixafor (AMD3100). We also used several concentrations of busulfan. We hypothesized that higher concentrations of busulfan and BMDC mobilization would increase numbers of GFP+ cells in the CNS. The doses of busulfan employed (60–125 mg/kg) all resulted in high levels of sustained chimerism (>85% 1 year post-transplant) in both the blood and BM of wild-type (WT) mice and an amyotrophic lateral sclerosis (ALS) mouse model. Moreover, cells accumulated within the CNS in a dose-, time-, and disease-dependent manner. Conditioning with the hydrophilic busulfan analog treosulfan, which is unable to cross the BBB efficiently, also resulted in a high degree of BM chimerism. However, few GFP+ BMDCs were found within the CNS of WT or ALS mice of treosulfan-conditioned mice. Mobilization of BMDCs into the circulation using GCSF and/or AMD3100 did not lead to increased accumulation of GFP+ BMDCs within the CNS of WT or ALS mice. Weekly analysis of BMDC accumulation revealed that BMDCs accumulated more rapidly and to a greater extent in the CNS of ALS mice conditioned with a high dose (125 mg/kg) of busulfan compared to a lower dose (80 mg/kg). The number of GFP+ BMDCs in the CNS labeling with the proliferation marker Ki67 increased in parallel with BMDC accumulation within the CNS. Our results indicate that establishment of high levels of blood and BM chimerism alone is not sufficient to induce BMDC accumulation within the CNS and that CNS conditioning is a crucial requirement for BMDC accumulation to occur. Moreover, it appears that proliferation of BMDCs that infiltrate the CNS is partly responsible for cell accumulation in busulfan-conditioned ALS mice.

Busulfan and melphalan versus carboplatin, etoposide, and melphalan as high-dose chemotherapy for high-risk neuroblastoma (HR-NBL1/SIOPEN): an international, randomised, multi-arm, open-label, phase 3 trial

High-dose chemotherapy with haemopoietic stem-cell rescue improves event-free survival in patients with high-risk neuroblastoma; however, which regimen has the greatest patient benefit has not been established. We aimed to assess event-free survival after high-dose chemotherapy with busulfan and melphalan compared with carboplatin, etoposide, and melphalan. We did an international, randomised, multi-arm, open-label, phase 3 cooperative group clinical trial of patients with high-risk neuroblastoma at 128 institutions in 18 countries that included an open-label randomised arm in which high-dose chemotherapy regimens were compared. Patients (age 1-20 years) with neuroblastoma were eligible to be randomly assigned if they had completed a multidrug induction regimen (cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide with or without topotecan, vincristine, and doxorubicin) and achieved an adequate disease response. Patients were randomly assigned (1:1) to busulfan and melphalan or to carboplatin, etoposide, and melphalan by minimisation, balancing age at diagnosis, stage, MYCN amplification, and national cooperative clinical group between groups. The busulfan and melphalan regimen comprised oral busulfan (150 mg/m2 given on 4 days consecutively in four equal doses); after Nov 8, 2007, intravenous busulfan was given (0·8-1·2 mg/kg per dose for 16 doses according to patient weight). After 24 h, an intravenous melphalan dose (140 mg/m2) was given. Doses of busulfan and melphalan were modified according to bodyweight. The carboplatin, etoposide, and melphalan regimen consisted of carboplatin continuous infusion of area under the plasma concentration-time curve 4·1 mg/mL per min per day for 4 days, etoposide continuous infusion of 338 mg/m2 per day for 4 days, and melphalan 70 mg/m2 per day for 3 days, with doses for all three drugs modified according to bodyweight and glomerular filtration rate. Stem-cell rescue was given after the last dose of high-dose chemotherapy, at least 24 h after melphalan in patients who received busulfan and melphalan and at least 72 h after carboplatin etoposide, and melphalan. All patients received subsequent local radiotherapy to the primary tumour site followed by maintenance therapy. The primary endpoint was 3-year event-free survival, analysed by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01704716, and EudraCT, number 2006-001489-17. Between June 24, 2002, and Oct 8, 2010, 1347 patients were enrolled and 676 were eligible for random allocation, 598 (88%) of whom were randomly assigned: 296 to busulfan and melphalan and 302 to carboplatin, etoposide, and melphalan. Median follow-up was 7·2 years (IQR 5·3-9·2). At 3 years, 146 of 296 patients in the busulfan and melphalan group and 188 of 302 in the carboplatin, etoposide, and melphalan group had an event; 3-year event-free survival was 50% (95% CI 45-56) versus 38% (32-43; p=0·0005). Nine patients in the busulfan and melphalan group and 11 in the carboplatin, etoposide, and melphalan group had died without relapse by 5 years. Severe life-threatening toxicities occurred in 13 (4%) patients who received busulfan and melphalan and 29 (10%) who received carboplatin, etoposide, and melphalan. The most frequent grade 3-4 adverse events were general condition (74 [26%] of 281 in the busulfan and melphalan group vs 103 [38%] of 270 in the carboplatin, etoposide, and melphalan group), infection (55 [19%] of 283 vs 74 [27%] of 271), and stomatitis (138 [49%] of 284 vs 162 [59%] of 273); 60 (22%) of 267 patients in the busulfan and melphalan group had Bearman grades 1-3 veno-occlusive disease versus 21 (9%) of 239 in the carboplatin, etoposide, and melphalan group. Busulfan and melphalan improved event-free survival in children with high-risk neuroblastoma with an adequate response to induction treatment and caused fewer severe adverse events than did carboplatin, etoposide, and melphalan. Busulfan and melphalan should thus be considered standard high-dose chemotherapy and ongoing randomised studies will continue to aim to optimise treatment for high-risk neuroblastoma. European Commission 5th Framework Grant and the St Anna Kinderkrebsforschung.

366 - Pharmacoeconomic Analysis and Clinical Outcomes of Staggered PK-Based Busulfan Dosing during Myeloablative Conditioning for Allogeneic Stem Cell Transplant Recipients

366 - Pharmacoeconomic Analysis and Clinical Outcomes of Staggered PK-Based Busulfan Dosing during Myeloablative Conditioning for Allogeneic Stem Cell Transplant Recipients