Busulfan-cyclophosphamide versus cyclophosphamide-busulfan as conditioning regimen before allogeneic hematopoietic cell transplantation: a prospective randomized trial

Busulfan and cyclophosphamide (BuCy) is a frequently used myeloablative conditioning regimen for allogeneic hematopoietic cell transplantation (allo-HCT). Theoretical considerations and pharmacological data indicate that application of busulfan prior to subsequent cyclophosphamide (BuCy) may trigger liver toxicity. Reversing the order of application to cyclophosphamide-busulfan (CyBu) might be preferable, a hypothesis supported by animal data and retrospective studies. We performed a prospective randomized trial to determine impact of order of application of Bu and Cy before allo-HCT in 70 patients with hematological malignancy, 33 patients received BuCy and 37 CyBu for conditioning. In the short term, there were minimal differences in liver toxicity favoring CyBu over BuCy, significant only for alanine amino transferase at day 30 (p = 0.03). With longer follow-up at 4 years, non-relapse mortality (6% versus 27%, p = 0.05) was lower and survival (63% versus 43%, p = 0.06) was higher with CyBu compared to BuCy. Other outcomes, such as engraftment (p = 0.21), acute and chronic graft-versus-host disease (p = 0.40; 0.36), and relapse (p = 0.79), were similar in both groups. We prospectively show evidence that the order of application of Cy and Bu in myeloablative conditioning in allo-HCT patients has impact on outcome. Electronic supplementary material The online version of this article (10.1007/s00277-020-04312-y) contains supplementary material, which is available to authorized users.

.1 Therapy schedules ___________________________________________________________________________ 21 11.2 Handling, preparation and administration of CY and BU _________________________________ 22 11.3 Allogeneic HSCT _____________________________________________________________________________ 23 11.4 Supportive care ______________________________________________________________________________ 23 12 EVALUATIONS AND INVESTIGATIONS BEFORE, DURING AND AFTER TRIAL _______ 23 12.1 Pretreatment evaluations and procedures _________________________________________________ 23 12.2 Evaluations to be performed within 21 days before treatment ___________________________ 23 12.3 Evaluations prior to start of the conditioning regimen ___________________________________ 23 12.4 Evaluations during trial ____________________________________________________________________ 24 12.5 Evaluations after treatment ________________________________________________________________ 24 12.6

Objectives
The aim of this study is to test the hypothesis, that the order of application of Busulfan (BU) and Cyclophosphamide (CY) has an impact on toxicity after allogeneic Hematopoietic stem cell transplantation (HSCT) and that CY-BU reduces liver toxicity compared to BU-CY.

Primary endpoint
Liver toxicity at day 30, assessed as serum values of ASAT, ALAT, GGT, AP, and bilirubin.

Secondary endpoints
Incidence and severity of Veno-occlusive Disease (VOD).
Incidence and severity of acute Graft-versus-Host Disease (GVHD), by organ (skin, liver, gut) at day 30 and day 100 Organ toxicity (definitions of the Common Terminology Criteria for Adverse Events (CTCAE), version 4.0) at day 30 and day 100 Survival, relapse and non relapse mortality at day 30 and day 100

Trial design
Prospective randomized multicenter trial.

Selection of patients (most important criteria)
Patients planned for an allogeneic HSCT with myeloablative conditioning Age 18 -65 years Myeloid leukemia respectively related precursor neoplasms, or lymphoid neoplasms.

Trial duration
The inclusion of patients is planned to start in Q3 2012 and will stop after the inclusion of 36 patients in each arm, which is expected in Q2 2014.

Statistical considerations
The primary objective of the study is to estimate the difference in liver toxicity between the two study arms on day 30. The sample size estimation is based on the previous observational study and theoretical assumptions. Sample size was set to ensure at least 80 % power, 1 -β = 0.8, at a 6 INTRODUCTION AND BACKGROUND

Therapy background
Busulfan (BU) -Cyclophosphamide (CY) is an established myeloablative-conditioning regimen for allogeneic hematopoietic stem cell transplantation (HSCT). It has a long-standing track record in the treatment of patients with leukemia and some congenital disorders and its advantages and disadvantages are well described. The antileukemic and immunosuppressive efficacy of BU-CY is considered to be equivalent to or even better than total body irradiation (TBI) in combination with CY.
(1-3) Liver toxicity and hepatic veno-occlusive disease (VOD) are the most frequent early lifethreatening complications associated with this approach.(4-6) Erratic absorption of oral BU has long been considered as a key factor for severe liver toxicity. Monitoring of serum levels has been advocated as a tool to reduce toxicity. Later on, the introduction of intravenous BU offered the advantage of easier administration with predictable pharmacokinetics and better tolerance than oral BU. (7) Both approaches reduced incidence and severity of liver complications but major problems remained.

Mechanism of action(8)
BU is an alkylating agent that interferes with DNA replication leading cells to apoptosis. BU enters the cells by passive diffusion. In vitro studies of cultured bone marrow cells demonstrate that BU at low concentrations is particularly cytotoxic for granulocytes and less cytotoxic to cells giving rise to other blood elements (platelets, lymphocytes, etc.

Metabolism(8)
BU i.v. is thought to be metabolised in a comparable way from that of the oral formulation.
Therefore, information on metabolism can be obtained from oral BU investigations. BU metabolism has been studied most extensively in rats and the same metabolites were identified in human.
All metabolites arise from the initial conjugation of BU with glutathione in a glutathione S transferase (GST) catalysed reaction. The most active human GST catalysing this reaction is GSTA1. Three major metabolite peaks have been identified and none of them are thought to contribute significantly to either efficacy or toxicity. The overall safety profile BU is mainly based on data from patients treated within clinical trials and on published literature or on NCI, EMEA or Swissmedic annual reports.

Metabolism and mechanism of action(9)
CY is biotransformed principally in the liver to active alkylating metabolites by a mixed function microsomal oxidase system. These metabolites interfere with the growth of susceptible rapidly proliferating malignant cells. The mechanism of action is thought to involve cross-linking of tumor

Distribution(9)
Concentrations of CY metabolites reach a maximum in plasma 2 to 3 hours after an intravenous dose.
CY achieves concentrations in the cerebrospinal fluid approximately equal to those in plasma. Plasma protein binding of unchanged drug is low (20%) but some metabolites are bound to an extent greater than 60%.

Elimination(9)
The mean half-life is 6-9 hours. CY is eliminated primarily in the form of metabolites, but from 5% to 25% of the dose is excreted in urine as unchanged drug. Several cytotoxic and non-cytotoxic metabolites have been identified in urine and in plasma.

Drug-related adverse events(9)
Bone marrow depression, fluid retention, cardiomyopathy, nausea and emesis, diarrhea, hepatic dysfunction, dizziness, blurred vision, hemorrhagic cystitis (prevented by forced diuresis and uromitexan, above), infertility, alopecia, erythematous skin rash. Other rarely adverse events are reported in the Swissmedic reports for Endoxan®. (9) The overall safety profile CY is mainly based on data from patients treated within clinical trials and on published literature or on NCI, EMEA or Swissmedic annual reports. These theoretical considerations and pharmacological data indicate that application of BU may trigger liver toxicity of subsequent CY, and suggest that reverse order of CY-BU would be preferable.

Rationale for performing the trial
This was suggested several years ago in a mouse model (17) and in a non-randomized pediatric study of autologous HSCT where BU induced less liver toxicity when given as second drug. In the present study, we want now to prospectively compare the order of application and liver toxicity in patients given BU and CY to confirm the aforementioned pharmacological, experimental and clinical results.

Cytokines profiling
Conditioning with BU and CY induced higher levels of inflammatory cytokines. (17)

Pharmakogenomics (Polymorphisms analysis)
Most of the drugs used to treat cancer are metabolised by hepatic enzymes such as cytochrome P450 (CYP450) or Glutathione-S-Transferase (GST). These enzymatic pathways can be more or less active in The current hypothesis is that some functional polymorphisms of genes (GST/CYPs), which control important enzymes in BU/CY metabolism, contribute to the observed interindividual variability in pharmacokinetics of these drugs. This variability can hence predict the resistance as well as the toxicity from a drug in patients who have cancer. The pharmacokinetic profile of different drugs, which have a hepatic metabolism, can be dramatically modified by these polymorphisms. In addition to these the variations and /or expression of the genes coding for the proteins involved in BER pathway might have an influence on the efficacy of BU and CY.
Hence, the current study is aimed at investigating the genetic variants and/or expression of the GST/CYP450 and the base excision repair (BER) pathway genes for their potential as molecular markers for predicting the efficacy and outcome of myeloablative therapy with BU and CY. We would like to study as well the relationship between the pharmacokinetics and pharmacogenetics of busulfan and cyclophosphamide.

alpha GST levels and glutathione analysis
Plasma alpha GST will be measured using HEPKIT alpha (a quantitative enzyme immunoassay from Biotrin International, Dublin, Ireland). The test procedure is based on the sequential addition of sample, anti alpha GST enzyme conjugate and substrate to microassay wells coated with anti alpha GST. The resultant color intensity is proportional to the amount of alpha GST present in the sample.
Assay range is 0-40 microg/L, equivalent to 0-200 microg/L in samples diluted 1:5. HEPKIT alpha is highly specific and no cross reactivity has been observed with either mu or pi isoforms of GST. (24) GST is stable in serum and can be stored 15 months at -20°C. Glutathione will be measured using a spectrophometric/ colorimetric method.

Expression analysis of BER and GST pathway genes and their influence on outcome to treatment
The RNA extraction will be performed for all the samples at 9-12 months of storage point using Trizol reagent and will be converted to cDNA using high capacitance cDNA Archive kit (Applied Biosystems, Foster City, USA) and will be stored at -80°C till further anlaysis.
Gene expression analysis will be performed using SYBR green chemistry on a SteponePlus real time PCR system (Applied Biosystems, Foster City, USA) with gene specific primers designed for the genes involved in BER pathway, GST and other genes of DNA repair pathways to be playing a role in repair of DNA damage produced by BU and CY.

Study Aim
The primary study goal of this prospective randomized study is to test the hypothesis, that the order of application of BU and CY has an impact on toxicity after allogeneic HSCT and that CY-BU reduces liver toxicity compared to BU-CY. Reduced liver toxicity is considered as a prerequisite for reduced liver GVHD and improved survival.

Primary endpoint
Liver toxicity at day 30, assessed as absolute serum values of ASAT, ALAT, GGT, AP, bilirubin at day 30.

Cytokines measurement
In animal models could be shown that the conditioning regimen with BU-CY induced higher levels of liver enzymes and inflammatory cytokines in comparison to a conditioning regimen with CY-BU. (17) This observation is important, because the proinflammatory cytokines, particularly IL-2 and TNFalpha, may be one of the first steps in the development of acute graft-versus-host disease (GVHD). (18)(19)(20) To test this correlation between order of application of the conditioning regimen and the levels of proinflammatory cytokines as well as the correlation between levels of cytokines and development of acute GVHD, plasma samples will be collected at different time points. At the end of the study the plasma samples will be analyzed with an ELISA test for proinflammatory cytokines as described previously.

Pharmacogenomics (polymorphisms)
Most of the drugs used to treat cancer are metabolised by hepatic enzymes such as cytochrome P450 (CYP450) or GST. These enzymatic pathways can be more or less active in the drug's metabolism according to the given polymorphism of each patient (pharmacogenomics). The current hypothesis is that some functional polymorphisms of genes, which control important enzymes in BU and CY metabolism, contribute to the observed interindividual variability in toxicity after allogeneic HSCT.
The pharmacokinetic profile of different drugs, which have a hepatic metabolism, can be dramatically modified by these polymorphisms.
In order to gain further insight into the role of these polymorphisms and their ability to predict the clinical response parameters (graft rejection, relapse following bone marrow transplantation, liver toxicity, VOD) we propose to correlate all these values with GST polymorphisms.

TRIAL DESIGN, DURATION AND TERMINATION
The study is designed as a prospective randomized multicenter study comparing CY-BU (group B, experimental group) as conditioning before allogeneic HSCT for myeloid leukemia with BU-CY (group A, standard group). The inclusion of patients is planned to start in Q3 2012 and will stop after the inclusion of 36 patients in each arm (see statistical considerations), which is expected in Q2 2014. End of trial (last patient, last visit) is expected for Q2 2015. All patients will be followed up for 100 days after allogeneic HSCT.

Inclusion criteria
Patients planned to undergo an allogeneic HSCT with myeloablative conditioning Patients with a history of hepatitis might be included, if no contraindication for HSCT exists.
Patient must give written informed consent

Exclusion criteria
Indication other than myeloid leukemia respectively related precursor neoplasms, or lymphoid neoplasms.

HIV infection
Donor other than HLA-identical sibling or min. 10/10 matched unrelated donor Pregnant or lactating women Lack of written informed consent

Enrollment
Prior to enrollment, the following steps have to be performed: Check the eligibility criteria Obtain written informed consent Fill in the patient screening, enrollment and identification list Enrolment of study patients will be documented in the electronic data capture (EDC) system secuTrial®. Study personnel will be provided with an individual login and password to access the electronic data capture system

After enrollment
Allogeneic HSCT should be performed within 3 months from subject registration.

Randomization
Patients will be randomized (1:1) to receive BU-CY (group A, standard group) or CY-BU (group B, experimental group) as conditioning regimen for allogeneic HSCT.
Randomization will be performed online via the electronic data capture system (secuTrial®).

Stratification
Patients will be stratified for randomization by center and donor type (related vs. unrelated).

Therapy schedules
The BU-CY conditioning (standard group A) regimen consists of intravenous BU 0.8 mg/kg administered every 6 hours (total 16 doses) on days -8 to -4 followed by intravenous CY 60 mg/kg on days -3 and -2.
The CY-BU conditioning regimen (experimental group B) consisted of intravenous CY 60 mg/kg on days -8 and -7 followed by intravenous BU 0.8 mg/kg administered every 6 hours (total 16 doses) on days -6 to -2.

Handling, preparation and administration of CY and BU
For handling, preparation and administration refer to the products information. (8,9) SUVA guidelines on handling of cytostatics have to be followed. (25) CY have to be dissolved in 500 ml 5% glucose or 0.9% NaCl; BU have to be diluted in NaCl 0.9% to a final concentration of 0.5mg/ml for stability reasons, as described in the products information of Busilvex®.(8) A 24 hours delay should be implemented between the BU and CY resp. CY and BU to decrease the eventuality of interactions between the two drugs and to increase the safety.

Allogeneic HSCT
The allogeneic HSCT will be performed at day 0 per intravenous route in approximately 15-30 min according to the institutional guidelines of the different centers. The graft will be checked for cell count.

Supportive care
Supportive care will be according to standard procedures described in appendix 1B.

Pretreatment evaluations and procedures
Informed consent must be obtained before the first examination.

Evaluations to be performed within 21 days before treatment
Study subjects will be screened for eligibility before registration in database.
Locally documented leukemia diagnosis, incl. disease stage

Evaluations during trial
Physical examination at day 0, 30 and 100 including Karnofsky performance status(26) (s.

Evaluations after treatment
Follow-up information for survival status, further treatments and, if applicable, progression/relapse at day 30 and 100. All responses, changes in response status and progression/relapse have to be documented on the eCRF.
Other examinations are performed according to the institutional guidelines of the different centers.

Sampling for pharmacogenomics, and cytokines profiling
Sampling and processing details are specified in Appendix 8.

Adverse Reaction (AR):
All untoward and unintended responses to a study drug judged by sponsor-investigator as having a reasonable causal relationship to the study drug. The expression reasonable causal relationship means to convey in general that there is evidence or argument to suggest a causal relationship.

Serious Adverse Event (SAE) or Serious Adverse Drug Reaction (SADR):
A SAE includes any of the events listed in the

Suspected Unexpected Serious Adverse Reaction (SUSAR):
SUSARs are serious adverse reactions that are assessed as unexpected on the basis of the applicable Swiss product information, (8,9) and the European summary of product characteristics.(29)

Causality assessment of adverse events
Most adverse events and adverse reactions that occur in the study, whether they are serious or not, will be expected treatment-related toxicities due to the drugs used in this study. The assignment of the causality should be made by the investigator responsible for the care of the participant using the definitions in the table below.

Unrelated:
The AE is clearly not related to the trial treatment. The AE is completely independent of trial treatment and/or evidence exists that the event is definitely related to another etiology.

Unlikely
The AE is doubtfully related to the trial treatment. Temporal association between the AE and the trial treatment and the nature of the event is such that the trial treatment is not likely to have had any reasonable association with the observed illness/event (cause and effect relationship improbable but not impossible).

Possible
The AE may be related to the trial treatment. Less clear temporal association; other etiologies also possible.

Probable
The AE is likely related to the trial treatment. Clear-cut temporal association and a potential alternative etiology is not apparent.

Definitely
The AE is clearly related to the trial treatment. Clear-cut temporal association and no other possible cause.
Not assessable There is insufficient or incomplete evidence to make a clinical judgment of the causal relationship.

Handling of adverse events
Subjects with AEs will be treated appropriately. Abnormal laboratory values will be repeated until normal or until the abnormality can be explained and the subject's safety is not at risk. In cases of a medical emergency, treatment is available in house and includes the 24h-availability of the reanimation team and intensive care facilities. In case of a fatal SAE, investigator will provide Clinical Trial Unit (CTU) Basel all requested information, who will forward this information to the independent ethics committee within the legally fixed timeframes.

Medical follow-up of adverse events
The investigator will ensure that the subject receives medical follow-up as necessary until the condition has stabilized or returned to normal state, even if the period of the trial is over.

Data Safety Monitoring Committee (DSMC)
To ensure the safety of the participating study subjects a DSMC will review on the basis of the FDA's

SAE and SUSAR reporting
In case of an SAE, relationship to the study drug will be assessed by the investigator using the definitions mentioned above. The principle investigator (PI) ensures complete collection and documentation information concerning the SAE on a standard SAE form. Completes information will then be forwarded to CTU Basel who reports to the independent ethic committee and Swissmedic on behalf of the PI according to guidelines within 7 or 15 days.

Annual safety report
An annual safety report will be provided to the competent authority and to the independent ethics committee by CTU Basel.

End of study reporting
The PI will inform the independent ethics committee and Swissmedic when the study ends within 90 days or 15 days if the study is terminated early. A final report is sent to Swissmedic within six months of study termination by the investigator.

Statistical Methods and Data Analysis
Detailed methodology for summaries and statistical analyses of the data collected in this study will be documented in a statistical analysis plan. The analysis plan will be finalized before database closure and will be under version control at the Clinical Trial Unit, University Hospital Basel.

Analysis Data Sets
The all-subjects-randomized (ASR) set will be used as a safety set. The intention to treat (ITT) set consists of patients in the ASR without any major protocol deviation. Treatment will be assigned as indicated in the randomization list. If there is for whatever reason a difference between the applied procedure and the randomization list, a per protocol (PP) set will be defined for all patients in the ASR without any major protocol deviation. Treatment will be assigned according to the procedure the patient received. Detailed justification and data listings will be provided for patients who were

Patient demographics and baseline characteristics
Demographics and relevant baseline variables will be presented for the ASR set. Categorical data will be presented as frequencies and percentages. For continuous variables, the lower and upper quartile as well as the median will be presented.

Primary Objective
The primary objective of the study is to estimate the difference in ALT concentration (primary endpoint) between the two study arms on day 30. The difference in location will be estimated together with a confidence interval(31) and will be tested with a Wilcoxon Test. The analysis will be performed on the ITT set, which is restricted to patients with follow-up measurements of the ALTconcentration on day 20 or 30, or both. For patients with missing ALT-concentration on day 30, the missing measurement will be replaced by one before day 30.
Supportive analysis I: In addition, the difference in ALT-concentration together with a 95% confidence interval will be estimated using a linear regression model with the baseline value of the ALT-concentration as covariate.
Supportive analysis II: If a PP set was defined all analyses will be repeated on the PP set.

Interim Analysis
No interim analysis is planned.

Sample Size Estimation
Sample size was estimated to be able to test if there is a significant difference in ALT concentration between the experimental group CY-BU and the standard group BU-CY. Sample size calculation was based on data of 75 patients used for a retrospective analysis by Cantoni et al. (22), with a very similar objective as the planned prospective study. In the retrospective study, a reduction of the ALT concentration in the CY-BU group of 47.14 % was observed. Sample size was calculated with a semiparametric resampling method as suggested by Davison & Hinkley.(32) This allows at the same time to account non-parametrically for the pilot data set and parametrically for the treatment shift, θ.
Each sample size, n i=1,…., 21 = 40, …., 120, was evaluated by sampling 3333 times n i individual patients with replacement from the data after centering the data on the ALT-concentration in the BU-CY group. Half of the drop-out rate of 9 %, as observed in a retrospective analysis. Figure 1 presents how sensitive the sample size is with respect to the expected reduction of the ALT concentration.

Case report forms and reports
The reports are based on the EBMT MED-A and MED-B forms; in addition electronic case report forms (eCRF) specifically created for this trial are used for items not mentioned in the EBMT MED-A and MED-B forms. The monitor will collect a copy of the EBMT MED-A and MED-B forms.
Centers must use a patient screening, enrollment and identification list in order to allow identification of a patient and proper usage of initials. This list must be kept at the center in the investigator site file.

ETHICAL CONSIDERATIONS
This protocol was written, and the trial is to be performed in accordance with the Declaration of Helsinki, the Guidelines of Good Clinical Practice issued by ICH and Swiss regulatory authorities requirements.(33-36) Before planning to enter any patients into this trial, the investigator has to make sure that the trial has been approved by the local ethics committee and that their center has officially been opened by Swissmedic, if applicable. The local investigator is responsible for ensuring that the study will be conducted in accordance with the protocol, the ethical principles of the There should be no ethical conflicts. Patients will be either treated with the standard approach (group A) or with the novel approach (group B). There are some indications that the novel approach will be less toxic but there is no proof so far.

Informed consent and patient information
The informed consent procedure must conform to the guidelines on Good Clinical Practice issued by ICH(36) and Swissmedic. All patients will be informed of the aims and procedures of the trial, the possible adverse events, how to react in case an adverse event occurs, and possible hazards to which he/she will be exposed. They will be informed as to the strict confidentiality of their patient data, but they need to know that their medical records may be reviewed for trial purposes by authorized individuals other than their treating physician. An investigator must provide the patient with sufficient opportunity to consider whether or not to participate and minimize the possibility of coercion or undue influence. The information provided shall be in a language intelligible to the patient and may not include any content that appears to waive any of the patient's legal rights, or appears to release the investigator, the sponsor, or the institution from liability for negligence. It will be emphasized that participation is voluntary and that the patient is allowed to refuse further participation in the trial whenever he/she wants. This will not prejudice the patient's subsequent care. Informed consent shall be obtained on a written form approved by the local ethics committee and signed by the patient. The patient information as well as a copy of the signed and dated informed consent will be handed to the patient.

Premature withdrawal
Patients have the right to refuse further treatment for any reason and at any time. Patients who decide to withdraw from the trial should be asked whether they also want to withdraw their consent for their data to be used for the follow-up assessments. For the patient's security, a last examination should be performed.
Patients may be withdrawn at any time from trial treatment at the discretion of the investigator due to a serious adverse event, or based on any other relevant medical condition.

Insurance
The Sponsor will indemnify patients for damages they have suffered as participants in the trial. For this purpose, the Sponsor has taken out a special insurance for clinical trials.

Monitoring
The monitor will contact and visit the centers regularly. He/she will be allowed to inspect the various records of the trial in accordance with local requirements. All source documents must be accessible for monitoring. The monitor will maintain patient confidentiality. For this trial the expected average monitoring visit frequency is at least every 3 months during the treatment phase. This frequency may be adjusted based on the recruitment and the stage of the trial.
Before enrollment of the first patient, a trial initiation visit will take place. The objective of this visit is to meet the local staff involved in the conduct of the trial (including sub-investigators, research nurse, clinical research coordinator, pharmacist), to describe the main features of the protocol, the use of the case report forms, the practicalities of the trial and to distribute the trial-specific investigator site file (ISF). The initiation visit has to be documented by the monitor on the 'checklist for initiation visit'. During monitoring visits, 100% source data verification (SDV) will be performed for the first patient at a center. If no major discrepancies are found, SDV may be reduced and only the following data will be verified for every patient: Informed consent, Inclusion/exclusion criteria, Serious Adverse Events (SAEs), Serious Adverse Drug Reactions (SADRs), Primary endpoint .
In case of inadequate data quality, 100% SDV will be performed for further patients until acceptable data quality is again obtained. The monitors must provide all monitoring reports to the Trial Chairperson within 2 weeks from the visit.
At the end of the trial the monitor will make a study closing visit to all sites to ensure that all documentation is complete.
Details regarding the monitoring activities will be specified in the separate document Monitoring Plan.

Auditing/inspecting
Authorities have the right to perform inspections, and the Coordinating centers as well as the independent ethics committees have the right to perform on-site auditing during working hours upon reasonable prior notice. Source data must be accessible for inspection and auditing visits.

Archiving
The investigator is responsible for archiving of the Investigator's file (including the original signed informed consent forms of all participants) for at least 10 years after the end or the termination of the trial.

Quality assurance
Several procedures guarantee quality of trial conduct: Reviews of protocol and forms according to standard operating procedures Requirements for principal investigators for participation: signed and dated CV and trialspecific agreement Validation of database and statistical analysis Data will be entered in an electronic CRF. Computerized and manual consistency checks will be performed; in case of inconsistencies queries will be issued. Once all these documents of a site are submitted to the CTU, they will be forwarded to Swissmedic, if applicable.
The investigator will only be allowed to register patients into the trial after Swissmedic has approved the center (if applicable).

Record retention
The center will retain copies of the patient trial records (eCRF, MED-A and MED-B form patient informed consent statement, laboratory printouts, drug inventory logs, and all other information collected during the trial) and documentation until at least 10 years after the termination of the trial.
In the event that the investigator retires or changes employment, custody of the records may be transferred to another competent person who will accept responsibility for those records. Written notice of such transfer will be given to the CTU and the ethics committee. The CTU will notify the regulatory authorities.

Drug Accountability
Drug supplies, which will be provided by the hospital pharmacy, University Hospital Basel, will be kept in a secure, limited access storage area under the storage conditions appropriate for the study drug. The research site staff will maintain records of the product's delivery to the trial site, the inventory at the site, the use by each subject, and the return to the hospital pharmacy or alternative disposition of unused product(s). These records will include dates, quantities, batch/serial numbers, and expiry dates. The research site staff will maintain records that document adequately that the subjects were provided the doses specified by the study protocol and reconcile all investigational product(s) received from the hospital pharmacy. At the time of return to the hospital pharmacy, the research site staff must verify that no remaining supplies are in the investigator's possession.

Samples banking
Samples will be banked and kept for 20 years for future analyses in connection with polymporphisms or cytokines and conditioning regimen after allogeneic HSCT according to the SAMW guidelines.(39) The biobanking is described in detail in the "Biobankreglement" and the corresponding patient informed consent form. The plasma banking for cytokines profiling will be done in the University Hospital of Basel, Switzerland. The DNA Banking will be done in HUG Geneva University Hospital, Geneva, Switzerland.
The patient retains the right to have the sample material destroyed at any time by contacting the local investigator. However, already obtained data from this material can be used for intended analyses. The sponsor will be the exclusive owner of any data, discoveries, or derivative materials from the sample materials and is responsible for the destruction of the samples at the request of the research patient through the local investigator or at the end of the storage period. The local investigator will provide the principal investigator/ sponsor with the required trial and patient numbers (UPN) so that any remaining blood and any other components from the cells can be located and destroyed.
If a commercial product is developed from this research project, the sponsor will own the commercial product. The patient will have no commercial rights to such product and will have no commercial rights to the data, information, discoveries, or derivative materials gained or produced from the sample.
Any new analysis on these samples not planned in this protocol has to be approved by the steering committee,by the relevant ethics committees and by Swissmedic, if applicable.

Trial registration
The trial is registered in the NIH's ClinicalTrials.gov registry (NCT01779882).

Scientific amendment
Any amendment which may have an impact on the conduct of the trial, the potential benefit of the trial, or may affect patient safety, including changes of trial objectives, trial design, patient population, sample sizes, trial procedures, or significant administrative aspects must have been accepted by the SBST Board. Such an amendment is termed scientific amendment and must have the approval of the respective ethics committee and Swissmedic prior to implementation.

Safety amendment
A safety amendment is a special kind of scientific amendment, which is released when it is necessary to eliminate immediate hazards to trial participants. A safety amendment requires immediate implementation at local sites, before approval of local ethics committee and Swissmedic has been given.

Administrative amendment
Amendments including administrative changes such as minor corrections and/or clarifications that have no effect on the way the trial is to be conducted have to be submitted to the ethics committee.
Such an amendment is called administrative amendment and may be implemented with immediate effect. A letter of receipt of the ethics committee has to be forwarded to the CTU.

Funding
Baxter SA and Robapharm/Pierre Fabre SA support the study with unrestricted funding for the administrative costs.

PUBLICATION
The trial results will always be submitted for publication in a peer reviewed scientific journal regardless of the outcome of the trial -unless the trial was terminated prematurely and did not yield sufficient data for a publication. The final publication of the trial results will be written by the Trial Chairperson, the Co-Chairperson, the Principal Investigators and the Trial Statistician on the basis of the statistical analysis performed by the trial statistician. A draft manuscript will be submitted for review to all co-authors. Authors of the main manuscript will include the Trial Chairperson (first author), the Co-Chairperson, the Principal Investigators, the members of the steering committee, and the trial statistician. Others who have made a significant contribution to the trial may also be included as author, or otherwise will be included in the acknowledgement.
Authors of correlative manuscripts (e.g. results of translational research) will include the Trial Chairperson, the Co-Chairperson, the Principal Investigators, and those persons who have made a significant contribution to the published results.
Interim publications or presentations of the study may include demographic data, overall results and prognostic factor analyses, results for secondary endpoints, but no comparisons between randomized treatment arms for the primary endpoint may be made publicly available before the recruitment is discontinued.
Any publication, abstract or presentation based on patients included in this study must be approved by the Trial Chairperson and the Co-Chairperson. This is applicable to any individual patient or any subgroup of the trial patients. Such a publication cannot include any comparisons between randomized treatment arms or an analysis of any of the study endpoints unless the final results of the trial have already been published.

Copyright
The information contained in this protocol is copyright protected by the sponsor. This information is given for the needs of the trial and must not be disclosed to persons outside of the trial without prior written consent of the steering committee.

Confidentiality
Trial-related data of the patient will be provided in a codified manner to the CTU. A sequential unique patient number (UPN) will be attributed to each patient registered into the trial.
Identification of patients must be guaranteed at the center. In order to avoid identification errors, a center specific identification number and the UPN have to be provided on the eCRF. Use the patient screening, enrollment and identification list. Patient confidentiality will be maintained according to applicable legislation. Patients must be informed of, and agree to, data and material transfer and handling, in accordance with Swiss data protection law. All information concerning the trial drugs supplied by Baxter SA and Robapharm/Pierre Fabre SA in connection with this trial and not previously published is considered confidential and proprietary information.

GVHD prophylaxis
The GVHD prophylaxis is identical in both cohorts and consisted of i) Cyclosporine A: intravenous 3 mg/kg once daily or per oral 6 mg/kg daily divided in two dose) from day -3 ii) Intravenous methotrexate: 10 mg/m2 on day 1 and 6 mg/m2 on day 3 and 6. A rescue therapy with leucovorin intravenous 15 mg 24 and 30 hours after application of methotrexate will be also performed.
iii) An additional GVHD prophylaxis (e.g. ATG) can be performed according to the institutional guidelines of the different centers. Details will be documented on the CRF (MED-A Form).

Prophylaxis of central nervous system adverse reactions
All the patients will receive a prophylaxis for BU-related central nervous system adverse reactions with i) Lorazepam: 1 mg per oral three times daily started 12 hours before the first BU dose and stopped 24 hours after the last BU dose.

Prophylaxis of hemorrhagic cystitis
To prevent a hemorrhagic cystitis after application of CY patients will receive i) Hyperhydration: Patients will be hydrated with D5'NS (glucose 5% NaCl 0.45% + 20 mEq KCl/l + 5 mg Furosemide/l) iv at 200 ml/hr for 72 hrs beginning 2 hrs before the first CY dose. KCl will be further supplemented in case of hypokalaemia. An average urinary flow of at least 100 ml/hr will be maintained during 48 hrs following the beginning of the CY infusion. Diuretics (e.g. furosemide) will be added during this period depending on fluid in-and output status.
iii) Sodium bicarbonate for urine alkalinization can be accessorily administrated: e.g. NaBic 8.4% intravenous 100 ml at the days with CY application and the following day (from day -3 resp. day -7 until day -1 resp. day -5 in the group A resp. B). iv) Uromitexan 300 mg/m2 will be administered at -10 min prior to CY infusion, + 4 hrs, +8 hours and +12 hours following CY infusion on days -3 and -2 in the group A resp. on days -7 and -6 in the group B.

Antiemesis
Before Busulfan and Cyclophosphamide infusions, patients will be premedicated with antiemetics according to the institutional guidelines of the different centers.

Allogeneic SCT
Allogeneic SCT will be carried out according to the standard guidelines and general operational procedures in the local allogeneic bone marrow transplantation centers. Other basic transplant strategies as well as supportive care measures are performed according to the institutional guidelines of the different centers. Details will be documented on the CRF (MED-A Form).

Special management orders
All men and pre-menopausal women should use adequate contraception during the study. Sperm should be frozen before the start of treatment from men who wish to have children.   I understand that any violation of the protocol may lead to early termination of the trial at my institution.
I agree to report to the CTU and the relevant ethics committee, within one working day, any clinical adverse event that is serious (SAE) and Serious Adverse Drug Reactions (SADRs), whether considered treatment-related or not. Follow-up reports will be provided within the timelines specified on the SAE Form.
I agree to keep accurate records on all patient information (case report forms and patient informed consent statement), and all other information collected during the trial for a minimum period of 10 years after termination of the trial.
I will provide the required documents and information for monitors and all people responsible for auditing/inspecting. I confirm to be present at the center and responsible for the whole trial period. In case I leave the center, I will take care to hand over the responsibility of the principal investigator at the center to somebody else and I will inform the CTU and the ethics committee accordingly.
I agree not to close the trial at my center without prior written information to the CTU.
I agree not to publicize all or any part of the results of the trial carried out under this protocol before primary publication in a peer-reviewed journal, and I agree to notify the steering committee of any planned publications thereafter.
I will take care that all members of the local trial team will comply with the content of this agreement.

Principal investigator:
Name:____________________________________________________ Title:__________________ Center:___________________________________________________________________________ Date:_________________________ Signature:__________________________________________ The signed original of this agreement has to be sent to the CTU. A copy of this page has to be submitted to your local ethics committee and another has to be stored in your investigator site file.

Samples shipping
If necessary (Basel and Zürich), the samples will be shipped at the end of the study to the laboratory in Geneva for the DNA analysis. The shipping will be organized by the CTU Basel.

DNA banking
The genetic polymorphism analysis and DNA banking (see also 14.3) will be done at the HUG Geneva University Hospital, Geneva, Switzerland.

Handling and processing of samples
For expression analysis of BER and GST pathway genes blood samples (in PAXgene® Blood RNA tube) tubes) will be collected either by direct venipuncture, via indwelling cannula inserted in a forearm vein, or via an indwelling intravenous access device such as a Broviac catheter or a Port-à-Cath according to the Appendix 9 "Checkliste for collection of samples for pharmacogenomics and cytokines".
No tubes will be provided by the sponsor, all the centers have to use their own material.

Samples shipping
If necessary (Basel and Zürich), the samples will be shipped at the end of the study to the laboratory in Geneva for the pathway analysis. The shipping will be organized by the CTU Basel.

Procedure for alpha GST levels analysis and glutathione analysis
Handling and processing of samples For alpha GST levels analysis blood samples (in EDTA tubes) will be collected either by direct venipuncture, via indwelling cannula inserted in a forearm vein, or via an indwelling intravenous access device such as a Broviac catheter or a Port-à-Cath according to the Appendix 9 "Checkliste for collection of samples for pharmacogenomics and cytokines".
No tubes will be provided by the sponsor, all the centers have to use their own material.

Samples shipping
If necessary (Basel and Zürich), the samples will be shipped at the end of the study to the laboratory in Basel for the alpha GST level analysis. The shipping will be organized by the CTU Basel.