Relapse and regimen-related toxicities remain major challenges in achieving long-term survival, particularly among older patients with high-risk myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous studies have demonstrated the feasibility of treosulfan-based conditioning, noting stable engraftment and low non-relapse mortality (NRM) in patients undergoing HLA-matched allo-HSCT. However, data on treosulfan-based conditioning in the HLA-haploidentical transplantation (HaploT) setting are limited. We retrospectively compared conditioning with fludarabine-cyclophosphamide (FC)-melphalan (110 mg/m2) and FC-treosulfan (30 g/m2) prior to HaploT using post-transplantation cyclophosphamide (PTCy) in patients with high-risk MDS/AML patients ≥ 50 years, transplanted from 2009-2021 at our institution (n = 80). After balancing patient characteristics by a matched-pair analysis, we identified twenty-one matched pairs. Two-year OS and LFS were similar among the groups (OS 66% and LFS 66%, p = 0.8 and p = 0.57). However, FC-melphalan was associated with a significantly lower probability of relapse compared to FC-treosulfan (0% vs. 24%, p = 0.006), counterbalanced by a higher NRM (33% vs. 10%, p = 0.05). Time to engraftment and incidences of acute and chronic graft-versus-host disease (GvHD) did not differ significantly. In conclusion, HaploT using FC-treosulfan in combination with PTCy in patients aged ≥50 years with MDS/AML appears safe and effective, particularly in advanced disease stages. We confirm the favorable extramedullary toxicity profile, allowing for potential dose intensification to enhance antileukemic activity.

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.

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.

BACKGROUND: The aims of this study are to report our experience with treosulfan-based conditioning regimens for patients with non-malignant hematologic conditions, correlating clinical outcomes at different time points post-transplant with treosulfan exposure (AUC).
METHODS: This study was a single-center observational study investigating overall survival (OS), disease-free survival (DFS), and event-free survival (EFS) end-points post-transplant. The consequences of treosulfan AUC with respect to toxicity, correction of underlying disease, and long-term chimerism were also explored using pharmacokinetic analysis.
RESULTS: Forty-six patients received 49 transplants with treosulfan and fludarabine-based conditioning between 2005 and 2023. Twenty-four patients also received thiotepa. Donor chimerism was assessed on either whole blood or sorted cell lines at different time points post-transplant. Thirty-nine patients received treosulfan pharmacokinetic assessment to evaluate cumulative AUC, with five infants receiving real-time assessment to facilitate daily dose adjustment. OS, DFS, and EFS were 87%, 81%, and 69%, respectively. Median follow-up was 32.1 months (range 0.82-160 months) following transplant. Lower EFS was associated with patient age (<1 year; p = .057) and lower cumulative treosulfan dose (<42 g/m2; p = .003). Stable donor chimerism in B-cell, NK-cell, and granulocyte lineages at 1-year post-transplant were more prevalent in patients receiving thiotepa conditioning. Two infants required daily dose adjustment to treosulfan to avoid high AUC.
CONCLUSIONS: Excellent clinical outcomes and stable chimerism were observed in this patient series. The addition of thiotepa conferred no significant toxicity and trended toward sustained ongoing donor engraftment. Correlating treosulfan AUC with long-term patient outcomes is required.

BACKGROUND: Type of conditioning regimen impacts the outcome of patients who undergo allogeneic HSCT since graft versus host disease (GVHD), infections, regimen related toxicities (RRT) are important causes of post-transplant mortality. Despite the RRT profile of busulfan, it is frequently used worldwide. Treosulfan has advantages in terms of dose of administration, lower incidence of sinusoidal obstruction syndrome and lower neurotoxicity. We retrospectively investigated outcomes of patients who underwent allogeneic HSCT with treosulfan or busulfan based conditioning regimens in our institution.
METHODS: Treosulfan was administered to 94 patients while 85 patients received busulfan. Our outcomes were RRT, chronic and acute GVHD, relapse related mortality (RRM), non-relapse mortality, and fungal infection. The clinical follow up data, regarding the primary and secondary endpoints of our study, of the patients who received treosulfan or busulfan based conditioning regimens were statistically analyzed.
RESULTS: The median follow-up was 14 months for the treosulfan group while it was 11 months for the busulfan group (p = 0.16). RRT was 11.7% and 7.1% for treosulfan and busulfan respectively. The incidence of extensive chronic GVHD was less frequent in the treosulfan group compared to the busulfan group (15.7% vs. 32.1%) (p < 0.001). The incidence of acute GVHD (Grade 3 or higher) was 32.2% in the treosulfan group while it was 31.6% in the busulfan group. The RRM was 17% in the treosulfan group while it was 34% in the busulfan group. The non-relapse mortality was 35.5% and 29.4% in the treosulfan group and in the busulfan group respectively (p = 0.962).
CONCLUSIONS: Treosulfan, with a lower RRM, lower chronic GVHD incidence and with a similar RRT profile appears to be a safe alternative to busulfan.

Treosulfan has shown promise in allogeneic hematopoietic cell transplantation (HCT) for its myeloablative properties and low toxicity. In this single-center retrospective propensity score-matched cohort study we compared treosulfan- and busulfan-based conditioning in allogeneic HCT for patients with myelodysplastic syndrome (MDS). This study included 138 adults who underwent allogeneic HCT for MDS or chronic myelomonocytic leukemia at Princess Margaret Hospital, Toronto, from 2015 to 2022. Using propensity score matching, we compared transplant outcomes between 2 well-matched cohorts who received conditioning with either fludarabine-treosulfan (FT) (n = 46) or fludarabine-busulfan with total body irradiation (FBT200) (n = 92). A scoring system based on patient age, Karnofsky performance score, and hematopoietic cell transplant comorbidity index was used to assign patients based on fitness to low-dose (30 g/m2) or high-dose (42 g/m2) treosulfan: 32 (69.6%) received high-dose treosulfan. The racial composition of the 2 groups was similar, with 27.2% and 21.7% of FBT200 and FT recipients, respectively, identifying as non-Caucasian (P = .61). Primary outcomes were analyzed at a median follow-up of 747 days. Of all participants, 116 (84.0%) received graft-versus-host disease (GVHD) prophylaxis with post-transplant cyclophosphamide (PTCY) and antithymocyte globulin (ATG). Patients who received FT had a superior 2-year overall survival (OS) compared to those who received FBT200: 66.9% (95% confidence interval (CI): 46.1 to 81.2) versus 44.5% (95% CI: 34 to 54.4), hazard ratio (HR): 0.43, 95% CI: 0.22 to 0.84 (P = .013). In multivariate analysis (MVA), only the use of fresh grafts (P = .02) and FT (P = .01) were associated with improved OS. FT was associated with superior 2-year relapse-free survival (RFS) compared to FBT200: 63.1% (95% CI: 42.6 to 77.9) versus 39.1% (95% CI: 29.1 to 49.1), HR: 0.44 (95% CI: 0.24 to 0.81), P = .008. In MVA, the use of fresh grafts (P = .03) and FT (P = .009) were associated with improved RFS. Recipients of FT demonstrated superior 2-year graft-versus-host disease relapse-free survival (GRFS) compared to those who received FBT200: 57.4% (95% CI: 37.8 to 72.8) versus 35.1% (95% CI: 25.5 to 45). In MVA, only FT was associated with superior GRFS (P = .02). FT recipients exhibited markedly superior 1-year event-free survival compared to recipients of FBT200 in univariate analysis (40.3% (95% CI: 25.9 to 54.2) versus 9.2% (95% CI: 4.4 to 16.3), HR: 0.47 (95% CI: 0.30 to 0.72), P < .001) and MVA (P = .004). FT was associated with lower 1-year nonrelapse mortality compared to FBT200 in univariate analysis (9.9% (95% CI: 3.0 to 21.8) versus 29.7% (95% CI: 20.6 to 39.3), HR: 0.41 (95% CI: 0.17 to 0.96), P = .04) and MVA (P = .04). Our study utilized propensity score matching to demonstrate superiority of treosulfan- over busulfan-based conditioning in stem cell transplantation of patients with MDS and is the first to evaluate the performance of treosulfan-based conditioning in combination with ATG and PTCY. As such, it contributes to the increasing body of evidence supporting the safety of treosulfan, even at the dose of 42 g/m2.

Patients undergoing hematopoietic stem cell transplantation (HSCT) for hematologic malignancies during childhood have an increased risk of developing long-term sequelae that are in part attributable to the conditioning regimen. The present study aimed to assess the occurrence of long-term toxicities in a population of children who underwent HSCT for hematologic malignancies using either treosulfan or busulfan in the conditioning regimen. The cumulative incidences of growth impairment, altered gonadal function, altered thyroid function, cataracts, secondary malignant neoplasia, and altered pulmonary function were evaluated retrospectively by univariable and multivariable analyses in a population of 521 pediatric patients with acute leukemias or myelodysplastic syndromes treated in 20 Italian transplant centers affiliated with the Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP). The median duration of follow-up for the entire study population was 7.1 years (range, 1 to 16 years). Overall, a larger proportion of patients given busulfan developed long-term toxicities compared to patients treated with treosulfan (34% versus 20%; P = .01). In univariable analysis, gonadal toxicity developed in 10% of patients who received treosulfan (95% confidence interval [CI], 3% to 15%), compared with 38% (95% CI, 24% to 39%) of busulfan-treated patients (P = .02), and this finding was confirmed by multivariable analysis (relative risk, .51; 95% CI, .34 to .76; P = .0009). We did not find any statistically significant associations between the occurrence of other long-term toxicities and the use of either busulfan or treosulfan. This study provides evidence that the use of treosulfan is correlated with a reduced incidence of gonadal toxicity in children undergoing HSCT for hematologic malignancies.

Impaired testosterone secretion is a frequent sequela following hematopoietic stem cell transplantation (HSCT) in pediatrics, but long-term longitudinal trendlines of clinical and biochemical findings are still scanty.
Monocentric, retrospective analysis. Male patients transplanted <18 years between 1992 and 2021, surviving ≥2 years after HSCT and showing, upon enrollment, clinical and biochemical signs consistent with pubertal onset and progression were included. Clinical and biochemical data collected every 6-12 months were recorded.
Of 130 patients enrolled, 56% were prepubertal, while 44% were peri-/postpubertal upon HSCT. Overall, 44% showed spontaneous progression into puberty and normal gonadal profile, while the remaining experienced pubertal arrest (1%), isolated increase of FSH (19%), compensated (23%) or overt (13%) hypergonadotropic hypogonadism. Post-pubertal testicular volume (TV) was statistically smaller among patients still pre-pubertal upon HSCT (p 0.049), whereas no differences were recorded in adult testosterone levels. LH and testosterone levels showed a specular trend between 20 and 30 years, as a progressive decrease in sexual steroids was associated with a compensatory increase of the luteinizing hormone. A variable degree of gonadal dysfunction was reported in 85%, 51%, 32% and 0% of patients following total body irradiation- (TBI), busulfan-, cyclophosphamide- and treosulfan-based regimens, respectively. TBI and busulfan cohorts were associated with the lowest probability of gonadal event-free course (p<0.0001), while it achieved 100% following treosulfan. A statistically greater gonadotoxicity was detected after busulfan than treosulfan (p 0.024). Chemo-only regimens were associated with statistically larger TV (p <0.001), higher testosterone (p 0.008) and lower gonadotropin levels (p <0.001) than TBI. Accordingly, the latter was associated with a 2-fold increase in the risk of gonadal failure compared to busulfan (OR 2.34, CI 1.08-8.40), whereas being pre-pubertal upon HSCT was associated with a reduced risk (OR 0.15, CI 0.08-0.30).
a) patients pre-pubertal upon HSCT showed a reduced risk of testicular endocrine dysfunction, despite smaller adult TV; b) patients showed downwards trend in testosterone levels after full pubertal attainment, despite a compensatory increase in LH; c) treosulfan was associated to a statistically lower occurrence of hypogonadism than busulfan, with a trend towards larger TV, higher testosterone levels and lower gonadotropins.

Treosulfan is a structural analog of the alkylating agent busulfan which has been shown in clinical trials to exhibit comparable myeloablative activity while causing fewer serious side effects. Treosulfan is currently being considered for FDA approval in combination with fludarabine, one of the most commonly used myeloablative agents, as a conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Because plasma concentrations of both treosulfan and fludarabine exhibit significant interindividual variability, therapeutic drug monitoring (TDM) is indicated to ensure dosages are administered that maximize efficacy while minimizing toxicity. In this chapter, we describe a rapid, accurate assay to detect treosulfan and fludarabine simultaneously in human plasma using turbulent flow liquid chromatography coupled to electrospray ionization tandem mass spectrometry (TFLC-ESI-MS/MS). Treosulfan and fludarabine are extracted from only 100 μL of acidified plasma via protein precipitation with methanol containing isotope-labeled internal standards. The extract is injected into the TFLC-ESI-MS/MS system, and the analytes are quantified using multiple reaction monitoring and a six-point calibration curve.

Sickle cell disease affects the whole body through acute and chronic organ damage and results in significant physical and neurological constraints. The report by Cseh et al. demonstrates in a retrospective multinational study that allogeneic haematopoietic stem cell transplantation from HLA-identical siblings using a contemporary conditioning regimen is safe and effective in more than 96% of patients. Commentary on: Cseh et al. Busulfan-fludarabine- or treosulfan-fludarabine-based conditioning before allogeneic HSCT from matched sibling donors in paediatric patients with sickle cell disease: A study on behalf of the EBMT Paediatric Diseases and Inborn Errors Working Parties. Br J Haematol 2024;204:e1-e5. [Correction added on 23 November 2023, after first online publication: In the preceding sentence, the article title and doi have been updated in this version.].