Relapse remains the main cause of treatment failure in patients with myeloid malignancies even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We observed a particularly low incidence of relapse in patients prepared with fludarabine, busulfan and melphalan in our previous study and this multicenter retrospective analysis aimed to confirm the feasibility of the regimen and to identify the potential prognostic factors. This study was performed using registry data from adults patients with myeloid malignancies who underwent their first allo-HSCT following fludarabine(≥100 mg/m2), busulfan (≥3.2 mg/kg) and melphalan (≥100 mg/m2) based conditioning at nine transplantation centers in China between Jan. 2020 and Mar. 2022. A total of 221 consecutive patients (AML n = 171, MDS-IB-1 or 2 n = 44, CMML n = 6) with median age of 46 were enrolled in this study. The median follow-up was 507 days for survivors. The 2-year NRM, CIR, OS and DFS were 10.6% ± 2.2%, 14.8% ± 3.3%, 79.4% ± 3.7% and 74.6% ± 3.7%, respectively. In multivariate analyses, high HCT-CI (≥3) was the only independent factor for higher NRM [hazard ratio (HR), 2.96; 95% confidence interval (CI), 1.11 to 7.90; p = 0.030] and ECOG score ≥2 was the only independent factor for inferior OS (HR, 2.43; 95%CI, 1.15 to 5.16; p = 0.020) and DFS (HR, 2.12; 95%CI, 1.13 to 4.02; p = 0.020). AML diagnosis and positive measurable residual disease (MRD) at transplantation were predictors for higher CIR (HR = 7.92, 95%CI 1.05-60.03, p = 0.045; HR = 3.64, 95%CI 1.40-9.44, p = 0.008; respectively), while post-transplantation cyclophosphamide based graft-versus-host disease prophylaxis was associated with lower CIR (HR = 0.24 95%CI 0.11-0.54, p = 0.001). The intensity of conditioning regimen did not impact CIR, NRM, DFS and OS. These results supported that double alkylating agents of busulfan and melphalan based conditioning regimens were associated with low relapse rate and acceptable NRM in adult patients with myeloid malignancies. The optimal dose remained to be confirmed by further prospective studies.

UNASSIGNED: Humanized mouse models to recapitulate human biological systems still have limitations, such as the onset of lethal graft-versus-host disease (GvHD), a variable success rate, and the low accessibility of total body irradiation (TBI). Recently, mice modified with the CD47-SIRPA axis have been studied to improve humanized mouse models. However, such trials have been rarely applied in NOD mice. In this study, we created a novel mouse strain, NOD-CD47nullRag2nullIL-2rγnull (RTKO) mice, and applied it to generate humanized mice.
UNASSIGNED: Four-week-old female NOD-Rag2nullIL-2rγnull (RID) and RTKO mice pre-conditioned with TBI or busulfan (BSF) injection were used for generating human CD34+ hematopoietic stem cell (HSC) engrafted humanized mice. Clinical signs were observed twice a week, and body weight was measured once a week. Flow cytometry for human leukocyte antigens was performed at intervals of four weeks or two weeks, and mice were sacrificed at 48 weeks after HSC injection.
UNASSIGNED: For a long period from 16 to 40 weeks post transplantation, the percentage of hCD45 was mostly maintained above 25% in all groups, and it was sustained the longest and highest in the RTKO BSF group. Reconstruction of human leukocytes, including hCD3, was also most prominent in the RTKO BSF group. Only two mice died before 40 weeks post transplantation in all groups, and there were no life-threatening GvHD lesions except in the dead mice. The occurrence of GvHD has been identified as mainly due to human T cells infiltrating tissues and their related cytokines.
UNASSIGNED: Humanized mouse models under all conditions applied in this study are considered suitable models for long-term experiments based on the improvement of human leukocytes reconstruction and the stable animal health. Especially, RTKO mice pretreated with BSF are expected to be a valuable platform not only for generating humanized mice but also for various immune research fields.

Limited data on treosulfan pharmacokinetics in adults, particularly regarding autologous stem cell transplantation (ASCT) in acute myeloid leukemia (AML), is available to date. Furthermore, correlations between treosulfan exposure, toxicity, and clinical outcome remain understudied. In this single-center retrospective study, we analyzed data from 55 AML patients who underwent HDCT with treosulfan (14 g/m2) and melphalan (140 mg/m2 or 200 mg/m2) (TreoMel) between August 2019 and November 2023 at the University Hospital of Bern. We assessed treosulfan pharmacokinetics and correlations with several physiological parameters with potential impact on its interpatient variability. We further analyzed how treosulfan exposure correlates with toxicity and clinical outcomes. Women above 55 years showed higher area under the curve (AUC) levels (median: 946 mg*h/L, range: 776-1370 mg*h/L), as compared to women under 55 (median: 758 mg*h/L, range: 459-1214 mg*h/L, p = 0.0487). Additionally, women above 55 showed higher peak levels (median: 387 mg/L, range: 308-468 mg/L), as compared to men of the same age range (median: 326 mg/L, range: 264-395 mg/L, p = 0.0159). Treosulfan levels varied significantly with body temperature, liver enzymes, hemoglobin/hematocrit., and treosulfan exposure correlated with diarrhea severity in women over 55 (p = 0.0076). Our study revealed age- and gender-related variability in treosulfan pharmacokinetics, with higher plasma levels observed in female patients above 55. Moreover, our data suggest that treosulfan plasma levels may vary with several physiological parameters and that higher treosulfan exposure may impact toxicity. Our study underlines the need for further research on treosulfan pharmacokinetics, especially in older patients undergoing HDCT in the ASCT setting.

Several studies investigated the application of Mesenchymal stem cells (MSCs) for treating spermatogenic disorders. Considering the limitation of MSC application, the present study aimed to compare Wharton\'s jelly MSCs secretomes, including condition medium (CM) 10-fold concentrated (CM10), 20-fold concentrated CM (CM20), and extracellular vesicles (EVs) to restore busulfan-induced damage on male mice reproduction. So, Wharton\'s jelly MSCs were cultured, CM was collected, and EVs were isolated. Seventy-two mice were randomly assigned to nine groups, including Control, Busulfan 1 month (1M), Busulfan 2 months (2M), CM10, Busulfan + CM10, CM20, Busulfan + CM20, EVs, and Busulfan + EVs groups. Sperm characteristics, DNA maturity, DNA fragmentation index (DFI), and testicular gene expression were evaluated. Data analysis revealed that CM10 significantly improved sperm plasma membrane integrity, sperm DNA maturity, and DFI in the Busulfan + CM10 group compared to the Busulfan 2M group. Although CM20 and EVs showed a non-significant improvement. Gene expression analysis showed busulfan administration significantly decreased the expression of AR, CREB1, and PLCζ genes, while CM10 significantly restored CREB1 gene expression. The present study demonstrated that CM10 is more effective than CM20 or EVs in reducing busulfan-induced reproductive toxicity.

Busulfan exposure has previously been linked to clinical outcomes, hence the need for therapeutic drug monitoring (TDM). Study objective was to evaluate the effect of day 1 TDM-guided dosing (regimen d1) versus days 1 + 2 TDM-guided dosing (regimen d1 + 2) on attaining adequate busulfan exposure. In this observational study, we included all adults who received an allogeneic HCT with intravenous once daily busulfan over 4 days as part of the conditioning regimen at the University Medical Centre Utrecht or between July 31, 2014 and November 12, 2021. The primary outcome was attainment of the therapeutic busulfan target (cumulative area under the curve [AUCcum] 80-100 mg*h/L). Dose adjustment was based on the estimated AUC of the preceding dosing day(s). Additional TDM was performed in the event of large dose adjustments (≥25%). The choice of TDM regimen was solely based on the first day the busulfan dose was administered (regimen d1 + 2 occurred when conditioning started on a Saturday). In all patients, blood sampling was performed on day 4 for evaluation. The AUCcum was estimated using a validated population pharmacokinetic model. Busulfan target exposure was compared between both TDM regimen groups using a propensity score adjusted logistic regression model. The variance in the AUCcum between the TDM regimens was compared using the F-test. Patients were stratified for age (categorical). In regimen d1, 87.6% (n = 113/129) attained a therapeutic busulfan exposure, while in regimen d1 + 2 a proportion of 97.4% was found (n = 74/76, adjusted odds ratio for non-therapeutic AUC = 0.19, 95% confidence interval [95% CI]: 0.04-0.89). Variance of busulfan exposure in the regimen d1 group (SD = 6.8 mg*h/L) differed significantly from the variance in the regimen d1 + 2 group (SD = 3.6 mg*h/L, F-test, P < .001). Performing busulfan TDM on both day 1 and day 2, rather than only on day 1, improves busulfan target exposure attainment in adults undergoing HCT, provided that subsequent TDM is carried out if required.

BACKGROUND: Ewing sarcoma (ES), is a rare cancer affecting children, adolescents and adults. After VIDE (vincristine-ifosfamide-doxorobucin-etoposide) induction chemotherapy, Busulfan-Melphalan (BuMel) high-dose chemotherapy followed by autologous hematopoietic stem cells transplantation improved outcomes in unfavourable localized ES, but with more toxicities than conventional chemotherapy (VAI: Vincristine-dactinomycin-Ifosfamide). We evaluated whether the risk of acute toxicity associated with BuMel compared to VAI varied according to age in patients recruited in the R2Loc and R2Pulm randomised trials of the Euro-E.W.I.N.G.99 and Ewing-2008 trials.
METHODS: We included patients with a localized high-risk disease, or pulmonary or pleural metastasis. We analysed the risk of severe toxicity according to randomised treatment group (VAI versus BuMel) and age group (<12 years, 12-17 years, 18-24 years, ≥25 years). We evaluated the heterogeneity of treatment effects by age group using interaction terms in logistic multivariable models.
RESULTS: The analysis included 243 patients treated with VAI and 205 with BuMel. Overall, BuMel was associated with a higher risk of severe acute toxicity than VAI particularly haematological, gastrointestinal, liver, sinusoidal occlusive syndrome, and infections. Severe haematological toxicity and lower general condition were significantly more frequent in younger patients, whatever treatment. We did not observe any significant heterogeneity in terms of the excess risk of severe toxicities associated with BuMel compared to VAI according to age group.
CONCLUSIONS: The excess of acute toxicity associated with BuMel compared to VAI does not vary significantly with age, suggesting the feasibility of BuMel across all age groups.

UNASSIGNED: Since available therapeutic approaches for chemotherapy-induced non-obstructive azoospermia (NOA) patients are not enough efficient, an urgent need for treatment alternatives is felt. This study shows that adipose tissue-derived mesenchymal stem cells-derived exosome (AD-Exo) treatment is more effective in ameliorating busulfan-induced NOA rat models compared to platelet-rich plasma (PRP).
UNASSIGNED: Patients with non-obstructive azoospermia (NOA) are unable to have their children. Therefore, there is an urgent need for additional treatment alternatives for these patients. Recently, novel treatments based on the exosomes derived from mesenchymal stem cells (MSCs) as the agents responsible for exerting the paracrine effects and consequently biological functions of MSCs are proposed. Besides, platelet-rich plasma (PRP) as a significant blood byproduct has been therapeutically applied in several male infertility studies. In this study, we compared the effects of PRP and exosome treatment on spermatogenesis restoration in NOA rat models. Exosomes and PRP were isolated from the adipose tissue-derived MSCs (AD-MSCs) collected from conditioned medium and peripheral blood of human volunteers, respectively. Non-obstructive azoospermia (NOA) induction was done through two doses of busulfan at a 21-day interval. Thirty-five days after NOA induction, intratesticular injection of AD-MSCs-derived exosome (AD-Exo), PRP, and PBS was performed. The control group did not receive any treatment. Two months later, the rats were euthanized for further analysis. Our results revealed that both AD-Exo and PRP treatments improved the size and weight of testis, modulated the expression level of Dazl, Ddx4, Stra8, Pwil1, and Ccna1, and ameliorated the serum level of LDH, SOD, and GR enzymes in NOA rats. Moreover, the AD-Exo group showed improved testosterone, GPx, MAD, and CAT serum levels, sperm motility, and protein levels of DAZL and DDX4. This investigation verified the more efficient effects of AD-Exo treatment in comparison to PRP in ameliorating busulfan-induced NOA rat models.

UNASSIGNED: Oligospermia is one of the most common reasons for male infertility which is troubling numerous couples of child-bearing age. This investigation scrutinizes the implications and mechanistic underpinnings of ursolic acid\'s effect on busulfan-induced oligospermia in mouse models.
UNASSIGNED: A singular intraperitoneal injection of busulfan at a dosage of 30 mg/kg induced oligospermia. Two weeks subsequent to this induction, mice were subjected to various dosages of ursolic acid (10, 30, and 50 mg/kg body weight, respectively) on a daily basis for four consecutive weeks. Following this treatment period, a meticulous analysis of epididymal sperm parameters, encompassing concentration and motility, was conducted using a computer-assisted sperm analysis system. The histopathology of the mice testes was performed utilizing hematoxylin and eosin staining, and the cytoskeleton regeneration of the testicular tissues was analyzed via immunofluorescent staining. Serum hormone levels, including testosterone, luteinizing hormone, and follicle-stimulating hormone, as well as reactive oxygen species levels (inclusive of reactive oxygen species and malondialdehyde), were gauged employing specific enzyme-linked immunosorbent assay kits. Differentially expressed genes of testicular mRNA between the oligospermia-induced group and the various ursolic acid treatment groups were identified through RNA sequencing analysis.
UNASSIGNED: The results revealed that a dosage of 50 mg/kg ursolic acid treatment could increase the concentration of epididymal sperm in oligospermia mice, promote the recovery of testicular morphology, regulate hormone levels and ameliorate oxidative damage. The mechanism research results indicated that ursolic acid increased the expression level of genes related to motor proteins in oligospermia mice.

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

In addition to their immunosuppressive effect, cytostatics conditioning prior to adoptive therapy such as chimeric antigen receptor (CAR) T cells may play a role in debulking and remodeling the tumor microenvironment. We investigated in vitro the killing efficacy and impact of treosulfan and fludarabine on ovarian cancer cells expressing mesothelin (MSLN) and effect on MSLN-targeting CAR T cells. Treosulfan and fludarabine had a synergetic effect on killing of SKOV3 and OVCAR4 cells. Sensitivity to the combination of treosulfan and fludarabine was increased when SKOV3 cells expressed MSLN and when OVCAR4 cells were tested in hypoxia, while MSLN cells surface expression by SKOV3 and OVCAR4 cells was not altered after treosulfan or fludarabine exposure. Exposure to treosulfan or fludarabine (10 µM) neither impacted MSLN-CAR T cells degranulation, cytokines production upon challenge with MSLN + OVCAR3 cells, nor induced mitochondrial defects. Combination of treosulfan and fludarabine decreased MSLN-CAR T cells anti-tumor killing in normoxia but not hypoxia. In conclusion, treosulfan and fludarabine killed MSLN + ovarian cancer cells without altering MSLN-CAR T cells functions (at low cytostatics concentration) even in hypoxic conditions, and our data support the use of treosulfan and fludarabine as conditioning drugs prior to MSLN-CAR T cell therapy.