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.