Update of the study timelines due to delayed study start Clarification on withdrawal of patients who violate eligibility criteria prior to administration of IMP, i.e. withdrawals without IMP had to be replaced. To ensure that the inclusion/exclusion criteria remain fulfilled until start of conditioning the most current result had to be used for assessment of eligibility in case of repeated examinations. Exclusion criterion number 8 was revised. Clarification of the period in which the patient registration had to be performed. New safety information was added to the protocol and the patient information for parents. Reference to the document “Development Summary of Product Characteristics” for Treograft was added. A narrower therapeutic window range has been defined for a target AUCday0-4 in order to ensure a dose-adjustment of the full myeloablative dose in case Therapeutic drug monitoring of Busulfan has to be performed. The wording for the administration time of i.v. treosulfan and i.v. fludarabine was changed from “within” 120/30 minutes to “over” 120/30 minutes. In addition to prophylactic also therapeutic treatment of GvHD (eg,glucocorticoids) and anti-infective medication administered for anti-bacterial, antiviral and anti-fungal prophylactic treatment had to be documented on the CRF . Exceptions from expedited SAE/SAR reporting, clarification regarding documentation of laboratory parameters as adverse events, detailed instructions on SAE reporting and Instructions on documentation of “pregnancy” as SAE were added. A separate SAE report form has to be completed for each SAE. A change in the number of patients subjected to blood samples has been implemented in case an interim analysis reveals that the population PK model is not sufficiently accurate, or the DMC recommend dose modifications. The accepted time deviation of the first PK sample has been defined. “Graft failure” has been additionally considered as a competing event for aGvHD and cGvHD.

Changes in Sponsor personnel and job titles. Changes in timelines. Up-to-date information regarding safety was provided in the Investigator’s Brochure (approved edition 9.0, released 29-Nov-2015), including the Development SmPC for treosulfan, and in the SmPC for Busilvex. Safety information was deleted from the Clinical Trial Protocol. Additional blood samples were required for the validation of the POP-PK model. Based on a DMC recommendation dated 15-Apr-2016, PK sampling was to be performed until adequate PK data were available for each of the age groups. Editorial changes to improve readability were made and the sentences referring to the pooled statistical analysis and the interim analyses were moved. Subject Information Sheets were updated according to the above mentioned changes.

The CRO for regulatory aspects and/or monitoring outside of DE changed. In addition, the outsourcing was extended to all countries involved in the clinical trial and to project management. The Sponsor’s staff changed. In addition, a Medical Expert was named in order to provide a single point of contact to advise on trial related medical questions or problems. Functions of the International Coordinating Investigator and the National Coordinating Investigators were specified. The definition of engraftment after HSCT was clarified in order to avoid misinterpretations. The term “consecutive days” was defined as 3 consecutive blood samples, if these were taken on different days. The second and third samples were required to confirm stable engraftment above the defined thresholds and were to be taken on the next consecutive days whenever possible; however, exceptions were accepted. Update and submission of the Investigational Medicinal Product Dossier.

For particular non-malignant diseases like thalassaemia major or combined immunodeficiencies the reoccurrence of disease due to loss of donor cells may require the regular administration of blood products (eg, erythrocytes or PLT) as therapeutic agents. Thus, a return to transfusion dependence associated with the reoccurrence of the underlying disease after allo-HSCT was to be documented as rescue therapy on the CRF. Further, stem cell infusions with or without further conditioning regimens could be used as rescue therapy. A PIP modification was approved requesting to change the requirement to treat the vast majority of subjects (at least 85 out of 100) without additional use of thiotepa. Instead the unlimited use of the thiotepa containing therapy in qualified subjects was permitted. The last regular chimerism documentation (without signs of graft failure) was to be done 12 months after transplantation. The use of rescue therapy and graft failure was to be documented until the end of the longer-term follow-up phase. A donor type chimerism analysis was only required in order to confirm secondary graft failure after sustained decline of neutrophils (≤ 0.5 x 109/L) and leucocytes (≤ 1.0 x 109/L). In some non-malignant diseases like thalassaemia major, graft failure may not manifest as marrow aplasia, but as autologous reconstitution. In these cases, conventional definitions of graft failure based on the detection of cytopaenia could not be applied, but loss of chimerism was the sign of graft failure. Loss of chimerism in the cell compartment of interest led to recurrence of disease symptoms; loss of chimerism in the red cell compartment led to haemolysis and transfusion dependence in thalassaemia. In order to get clear evidence for a late secondary graft failure, the analysis of donor type chimerism was extended to the longer term follow-up phase. The definition of primary graft failure was specified as a donor-type chimerism of < 10% in BM.