Treosulfan–fludarabine–thiotepa-based conditioning treatment before allogeneic hematopoietic stem cell transplantation for pediatric patients with hematological malignancies

Treosulfan-based conditioning prior to allogeneic transplantation has been shown to have myeloablative, immunosuppressive, and antineoplastic effects associated with reduced non-relapse mortality (NRM) in adults. Therefore, we prospectively evaluated the safety and efficacy of treosulfan-based conditioning in children with hematological malignancies in this phase II trial. Overall, 65 children with acute lymphoblastic leukemia (35.4%), acute myeloid leukemia (44.6%), myelodysplastic syndrome (15.4%), or juvenile myelomonocytic leukemia (4.6%) received treosulfan intravenously at a dose of 10 mg/m2/day (7.7%), 12 g/m2/day (35.4%), or 14 g/m2/day (56.9%) according to their individual body surface area in combination with fludarabine and thiotepa. The incidence of complete donor chimerism at day +28 was 98.4% with no primary and only one secondary graft failure. At 36 months, NRM was only 3.1%, while relapse incidence was 21.7%, and overall survival was 83.0%. The cumulative incidence of acute graft-vs.-host disease was 45.3% for grades I–IV and 26.6% for grades II–IV. At 36 months, 25.8% overall and 19.4% moderate/severe chronic graft-vs.-host disease were reported. These data confirm the safe and effective use of treosulfan-based conditioning in pediatric patients with hematological malignancies. Therefore, treosulfan/fludarabine/thiotepa can be recommended for myeloablative conditioning in children with hematological malignancies.


Patient eligibility
Between November 2014 and July 2015, 70 children (aged 28 days to 17 years) with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or juvenile myelomonocytic leukemia (JMML) were enrolled in this prospective non-randomized phase II trial at 18 transplantation sites in five European countries. Written informed consent on all aspects of the study was obtained from all children and/or their legal guardians before enrollment. The study was approved by the responsible independent ethics committees and competent authorities and was performed in accordance with the Declaration of Helsinki, as well as Good Clinical Practice guideline. Safety and outcome parameters of all surviving patients were analyzed after a 36 months follow-up period focusing on the 65 patients who were additionally treated with thiotepa. The 5 out of 70 patients, who received treosulfan and fludarabine only, were excluded from the statistical analysis reported here. However, the key findings for all 70 trial patients (including the five patients conditioned only with treosulfan/fludarabine) are provided in Fig. S1 in the online supplementary.

Donors and grafts
Either human leucocyte antigens (HLA)-identical siblings (MSD), matched family donors, or matched unrelated donors were eligible donors (Table 1). An HLA match was defined as at least a 9/10 allele-matched after high-   resolution four-digit typing in HLA-A*, B*, C* and DRB1* and DQB1*.

Conditioning regimen and supportive care
All patients received an individualized BSA-adapted intravenous (IV) treosulfan dose on days −6 to −4, i.e., BSA of ≤0.5 m 2 received 10 g/m 2 /day; of >0.5-1.0 m 2 received 12 g/m 2 /day; and of >1.0 m 2 received 14 g/m 2 /day. Subsequently, fludarabine 30 mg/m 2 /day IV was given from day −7 to day −3 (total dose: 150 mg/m 2 ). At the investigator's discretion, thiotepa 2 × 5 mg/kg/day IV was additionally given on day −2 to 65 patients (total dose: 10 mg/kg). Allogeneic hematopoietic stem cells obtained either from peripheral blood or from bone marrow were given at day 0. Supportive care, including GvHD prophylaxis and treatment, was performed according to center-specific guidelines.

Endpoints and definitions
The objective of this phase II trial was to describe the safety and efficacy of treosulfan administered as part of a standardized fludarabine-containing conditioning treatment and to contribute to a PK model. Clinical endpoints included engraftment and complete donor-type chimerism (defined as ≥95% donor cells), NRM, disease relapse/progression (RI), relapse/progression-free survival (RFS/PFS), acute/chronic GvHD, GvHD-free and relapse/progression-free survival (GRFS) [27], cGvHD-free and relapse/progression-free survival (CRFS) [27], and overall survival (OS). NRM is defined as the probability of dying without previous occurrence of a relapse/progression of the underlying disease. Relapse/progression and graft failures are considered competing events. For estimation of GRFS, events were defined as acute GvHD of at least grade III, moderate or severe chronic GvHD, and relapse/progression or death (whichever occurred first). For CGRFS, events were defined as moderate or severe chronic GvHD, relapse/progression or death (whichever occurred first). All AEs except hepatic sinusoidal obstruction syndrome (HSOS) and hepatic toxicities were based on the Common Terminology Criteria for Adverse Events (CTCAE, version 4.03) and evaluated until 100 days after alloHSCT. HSOS was evaluated according to Jones et al. [28] and hepatic toxicities according to Bearman [29]. Furthermore, treosulfan concentrations in plasma from each patient were analyzed to calculate PK parameters in a subset of patients. Blood samples were taken immediately after treosulfan infusion and thereafter within 15-30 min, 1-2 h, 3-6 h, and 7-8 h. Further details of bioanalytical methods and the model-based parameter calculation have been previously described [26,30].

Statistical considerations
Descriptive analyses were performed using frequency, mean, standard deviation, median, and range, as appropriate. All time to event endpoints were measured from the day of HSCT (except for cGvHD, which started 100 days after HSCT) to the time of event or competing event, if applicable. Patients alive without event (or competing event) were censored at the last follow-up or at day +100 after HSCT for aGvHD. Kaplan-Meier estimates were used for calculating OS, GRFS, and CRFS. Cumulative incidences were used for estimating NRM, RI, aGvHD, and conditional cumulative incidences for neutrophil/leukocyte/ platelet engraftment. Competing events were as follows: relapse/progression and graft failure for NRM; deaths without relapse/progression and graft failure for RI; death, relapse/progression, and graft failure within 100 days after the end of HSCT for aGvHD; death, disease relapse/progression, or the use of rescue therapies for engraftment.
Any changes in single laboratory values were documented separately instead of being included in the AE analysis. Non-compartmental methodology was applied for the PK analysis of treosulfan. Based on the individual plasma concentration-time data, the following parameters were determined using the actual sampling times for treosulfan: C max , t max , AUC last , AUC ∞ , t 1/2 term, CL, and V d . Mean and standard deviations were calculated for all PK parameters, except for t max , for which the median and range were computed.
This trial was descriptive in nature, thus, p values (two sided) were explorative, based on a significance level of 0.1. The data were analyzed using Pearson chi-square test for chimerism, the log-rank test for OS, GRFS, and CRFS, and the Gray test for NRM, RI, a/cGvHD.
All analyses were predefined in the statistical analysis plan. SAS software (version 9.4; SAS, Cary, NC, USA) was used for statistical analyses. 6%] were conditioned with treosulfan, fludarabine, and thiotepa. Nearly all of the ALL and AML patients were in first or second complete remission and had received their first alloHSCT procedure. Patients with MDS were classified as having refractory anemia with excess blasts (50%) or refractory cytopenia (40%). Children received alloHSCT between November 2014 and July 2015.

Demographics
The median follow-up was 41.8 months (range for surviving patients: 24.2-57.5 months).

Outcomes
Engraftment, graft failure, and chimerism The number of patients achieving reconstitution of granulopoiesis was 64 (98.5%). One patient (1.4%) died before engraftment 15 days after alloHSCT. The maximum conditional cumulative incidence of engraftment reached was 100% (90% CI: 97.7, 100.0) 43 days after HSCT. No patient experienced primary graft failure, and one patient (1.4%) with ALL had decreased neutrophils and leukocytes counts, but presented 100% donor chimerism, for which the patient received a stem cell boost 113 days post transplant.
Non-relapse mortality and relapse/progression incidence Overall, the cumulative incidence of NRM at 36 months was 3.1% (90% CI: 0.0, 6.6). One patient with AML died due to laryngeal hemorrhage 0.5 months after HSCT. A second patient with MDS died due to multi organ failure 12.3 months after HSCT. Due to the low number of events, no statistically significant difference in NRM was detected among the three different treosulfan dose groups (Fig. 1a) or within any other of the analyzed subgroups.
The cumulative incidence of relapse/progression (RI) at 36 months was 21.7% (90% CI: 13.2, 30.1). In the subgroup analyses, no statistically significant impact was recorded for treosulfan dose, donor type, or patient's age group (Fig. 1b), but was found for underlying disease (p < 0.0001). Relapse incidences of ALL were 26.1%, AML 17.2%, MDS 0.0%, and JMML 100%. In the case of a 2nd HSCT procedure, the incidence of relapse/progression increased from 18.5 to 60.0% (p = 0.0140) considering that only five patients were treated in a second procedure.  (Table 3). There was no difference among the three different treosulfan dose groups (Fig. S2 in online supplementary).

Discussion
After initial data on treosulfan-based conditioning in adult alloHSCT patients became available [9,10], several published studies reported on the efficacy and safety of treosulfan given as part of various conditioning regimens in children [12,13,15,16,20,31]. Here, we report the final results of the first prospective alloHSCT trial of a treosulfan/fludarabine/thiotepa combination regimen (FTT; 65 of 70 trial patients treated) administered to children and adolescents with hematological malignancies. In this study, an individual BSA-adapted dose calculation was consistently applied for treosulfan. The 36-month Kaplan-Meier estimates of 3.1% for NRM, 83.0% for OS, and 73.6% for RFS/PFS compare favorably with the survival data reported for other conditioning regimens [2][3][4][5]. Overall, the 36-month relapse rate was low (21.7%), although three patients with JMML relapsed between 6 and 9 months post transplant. Disease recurrence is known to be particularly high in patients with JMML given an allograft, but the percentage of relapse seems to be higher than that reported using the busulfan/cyclophosphamide/ melphalan regimen [32]. However, due to the very low number of JMML patients, no conclusions can be drawn at this point in time. On the other hand, the 36-month relapse incidence of 26.1% for the ALL subgroup (23 patients) is probably comparable to that of standard TBI-based regimens. Chronic GvHD-free and relapse/progression-free survival Note: 'GvHD-free' defined as no acute GvHD of at least grade III and no moderate/severe chronic GvHD. 'Chronic GvHD-free' defined as no moderate/severe chronic GvHD. a Based on Kaplan-Meier estimates Fig. 3 Kaplan-Meier estimates of (a) GvHD-free and relapse/progression-free survival and (b) chronic GvHD-free and relapse/progression-free survival.
Numerically, it might be slightly higher compared with the data published by Peters et al. [2]. The value of TBI-based and chemotherapy-based conditioning in childhood ALL is currently being investigated in an international controlled phase III trial (EudraCT No.: 2012-003032-22; Clin-icalTrials.gov Identifier: NCT01949129). The relapse incidence of 17.2% at 36 months for the 29 AML patients and of 0% for the 10 MDS patients compares favorably with that reported for busulfan-based conditioning treatments [2,33,34]. Excellent results of the FTT regimen in children with AML (low relapse rate and reduced toxicity) warrant assessing this regimen in a prospective randomized pediatric alloHSCT trial in Europe, comparing busulfan-and treosulfan-based conditioning regimens for AML. All patients achieved neutrophil engraftment and 98.4% of patients had a complete donor chimerism by day +28 post transplant. While neutrophil engraftment was comparable to that of previous reports [12,20,31], no prospective trial data on chimerism have been previously reported in children with malignant disorders who received conditioning with FTT. Hence, this study provides confirmation of the myeloablative potential of a FTT conditioning regimen, which results in an excellent rate of complete donor-type chimerism.
There were no primary graft failures. One ALL patient developed secondary poor graft function with decreasing blood cell counts associated with a viral illness despite 100% donor chimerism and received a stem cell boost.
The incidence of liver toxicity was relatively low with only one case (1.5%) of grade II HSOS, which resolved under appropriate therapy, compared with an up to 22% HSOS incidence reported in busulfan-containing regimens [3,35]. Moreover, skin and subcutaneous tissue disorders of at least CTCAE grade III were within an acceptable range (12.3%) despite the intensification of the regimen with the addition of thiotepa. Just over half of the patients developed gastrointestinal (GI) AEs of at least CTCAE grade III, while oral mucositis was most prominent (43.1%). Compared with the data published by Boztug et al. [20], GI toxicity was relatively frequent, but not life-threatening, and was probably promoted by the addition of thiotepa in the vast majority of patients.
Unlike previous reports [22][23][24][25], we observed a rather limited variability in interindividual PKs of treosulfan. The BSA-banded dose calculation applied in this trial achieved equivalent treosulfan exposure (AUC, C max ) in all dose groups. In the dose subgroups, the differences in outcome parameters, such as overall survival, were therefore, probably not due to a higher treosulfan exposure, but may be related to specific patient-, graft-, and/or underlying disease characteristics. The wide age range of patients included in this trial (from 28 days to 17 years of age) is reflected by the increase in treosulfan plasma clearance and volume of   distribution within the different dose groups. Apparently, this observation is not related to treosulfan dose, but rather to the age-related differences in organ function maturation that are taken into account in the BSA-dependent dose calculation. Therefore, the strict application of the individual BSA-dependent treosulfan dose calculation should remain valid for the pediatric population [26]. There are several limitations to this prospective phase II study. Only a small number of patients were treated, especially in the MDS and JMML subgroups. Consequently, the poor outcome reported for JMML patients should be treated with caution. However, at least one JMML patient was considered at increased risk due to his second alloHSCT procedure.
The NRM, incidence of early toxicities, and donor chimerism, though, are likely to remain stable beyond this reported 36-month observation period. We can thus, safely conclude that FTT conditioning achieves high rates of engraftment with relatively low toxicity. Based on these and other clinical data, the European Commission has most recently approved treosulfan for conditioning in pediatric patients older than 1 month with malignant diseases [36].
We conclude that treosulfan-based conditioning with BSA-adapted dosing is safe and effective in pediatric patients with hematological malignancies. The cumulative incidences of OS and NRM compare favorably with those reported for other conditioning regimens. Treosulfan/fludarabine/thiotepa is, therefore, recommended as a suitable myeloablative preparative treatment for this pediatric patient population.
Acknowledgements The authors would like to thank the nursing staff and all parents/children who gave consent and participated in this trial. The trial was funded by medac GmbH (Wedel, Germany), the sponsor of the clinical phase II trial. The authors wrote the first draft of the paper, and all contributed to subsequent drafts and made the decision to submit the paper for publication. The principal and the corresponding authors had full access to patients' data and vouch for the veracity and completeness of the data and analyses (Funded by medac GmbH, MC-FludT.17/M, EudraCT No.: 2013-003604-39; Clinicaltrials.gov identifier: NCT02333058).

Compliance with ethical standards
Conflict of interest KK has received research funding from medac GmbH, Sanofi, and other travel grants. PB has received research funding and patents and royalties from medac GmbH, research funding from Riemser and Neovii. He also received compensation from Cellgene and Novartis for his consultations. In addition, he received compensation from Novartis for Speakers Bureau. BG received honoraria from Jazz Pharmaceuticals. KP received funding from medac GmbH and other funding for EBMT conference fees and accommodation. Prof. KW S received research funding and travel grants from medac GmbH and Aventis-Behring. SC received honoraria for his consultations from Gentium and Jazz Pharmaceuticals. RB received travel grants from medac GmbH and Neovii and is on Bluebird Bio's advisory board. FL received funding for EBMT/ASH conference fees and accommodation as well as speakers bureau from medac GmbH. AV is on the advisory board for Pfizer, medac GmbH, Novartis, Amgen, and Jazz Pharmaceuticals and also received other funding from them. CH, AKM, and JB are employees of medac GmbH. JB has licensed an employee's invention (PCT/EP00/10871) to medac GmbH.
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