Methods
This is a retrospective analysis performed at the Alberta Children’s Hospital (ACH) in Calgary, Canada. The inclusion criteria for the study were: age less than 18 years at time of transplant, a diagnosis of ALL requiring HSCT, first transplant, Bu/Flu/ATG + TBI conditioning regimen and transplant using bone marrow (BM) or peripheral blood stem cell (PBSC) products.
Institutional Review Board approval and waiver of consent was obtained prior to accessing the electronic records which spanned 17 years, from 2003 to 2020.
Conditioning
Prior to 2008 Bu was administered as an intravenous (IV) infusion on day -5 through -2 at a dose that was age determined (4mg/kg/dose in children less than four years of age and 3.2 mg/kg/dose if older) and without pharmacokinetic monitoring. Between 2008 and late 2010 pharmacokinetic monitoring was introduced and thereafter (In November 2010) a TDM dosing regimen was applied. In this regimen a test dose of Bu (25% of the actual dose) was administered on day -7. Serum drug levels were measured at end of infusion and 1, 3, 5 and 7 hours later. Based on the area under the curve (AUC) generated, the dosage of the Bu was adjusted. The first full dose of Bu was administered on day -5 and the drug levels were repeated to ensure adequate exposure to the drug. An AUC of 3750μmol*min was targeted across all Bu days including the test dose (total exposure 15000umol*min). Flu was administered on day -6 through -2 at a dose of 50 mg/m2 infused IV over one hour once . Rabbit - ATG (Thymoglobulin ® Sanofi Aventis) was given to all patients as a three-day course starting with 0.5 mg/kg on the first day (day -3) and weight-based dosing for the remaining two days (children over 30 kg received 2 mg/kg/dose and those less than 30kg received 2.5 mg/kg/dose, on days -2 to -1). Serum levels for ATG were not performed. Finally, 400 cGy TBI was given to all children in two divided doses on day -1.
Donor Sources
Donor and recipient human leukocyte antigen (HLA) matching was performed using the sequence based typing method until 2019 when next generation sequencing was introduced. HLA matching was done at 10 alleles for both PBSC and BM sources. Donors who were identical on all 10 alleles were considered matched, while those who were nine of ten were labelled mismatched. A haploidentical transplant was defined as any donor matching at five to eight out of 10 alleles.
GVHD prophylaxis and treatment
Cyclosporine was used for graft versus host disease (GvHD) prophylaxis in all subjects starting day -1 with dosage adjusted based on serum levels to target a therapeutic concentration between 150ng/ml and 200ng/ml. In addition, methotrexate 15 mg/m2 was administered on day 1 and 10mg/m2 on days 3, 6 and 11 post-transplant. Cyclosporine was subsequently weaned over five weeks starting on day 42 post-transplant in the absence of any GvHD.
Grading of GvHD was based on the modified Glucksberg criteria for acute graft versus host disease (aGvHD) and the National Institute of Health (NIH) criteria for chronic graft versus host disease (cGvHD).14,15 Once diagnosed, GvHD was managed as per institutional guidelines.
Supportive care
All patients received seizure prophylaxis during the administration of Bu and infectious prophylaxis with acyclovir, fluconazole, metronidazole and pentamidine (switched, after the first month, to sulfamethoxazole and trimethoprim). Cytomegalovirus (CMV) and Epstein Barr Virus (EBV) titres were monitored once weekly as were immunoglobulin G (IgG) levels. Chimerism testing was performed routinely on days 21 and 100 post-transplant and a level of donor cells more than 95% was considered complete donor chimerism.
Definitions and End points
In this study, the primary end point was Event Free Survival (EFS) at two years post-transplant. This was defined as the proportion of patients who remained alive and free from disease relapse and secondary malignancies. Overall survival (OS), defined as the proportion of subjects alive after the transplant as measured from the day of transplant to date of death or censored at day of last follow up, was also calculated.
Secondary end points included neutrophil engraftment which was defined as the first of three days with neutrophil count more than 500 cells/μl post-transplant, and platelet engraftment as the first day of platelets over 20,000 cells/ul for seven days without support with platelet transfusions.
The frequency and severity of GvHD (both and acute and chronic) were also calculated as were the rates of infections and SOS.
Statistical Analysis
For this analysis, we combined patients between 2008 and 2010 who met our assigned target AUC, with those transplanted after 2010 (who had Bu doses adjusted after pharmacokinetic monitoring). This targeted cohort was compared to an untargeted group. The untargeted group comprised of children who were transplanted before 2008 combined with those who had Bu measurements between 2008 and 2010 were outside the target range and were not corrected (Figure 1).
Patient characteristics and transplant data were described and proportions of the various characteristics between the Bu-targeted and untargeted groups. The two groups were compared using the Mann-Whitney U test for age at transplant and chi-square test for the nominal variables to detect the presence of any significant differences.
Neutrophil and platelet engraftment were described highlighting proportions of children successfully engrafting and the median time to engraftment. Cumulative rates of incidence of acute and chronic GvHD, viral infections and SOS were calculated using the Kaplan-Meier method. This analysis was also used to calculate the EFS and OS, cumulative incidences of relapse (CIR) and non-relapse mortality (NRM). The resultant values were compared using a log rank test between the Bu targeted and untargeted groups. Finally, a univariate analysis was performed to study the effect of variables on the EFS and OS. All statistical analysis was performed using SPSS version 26.