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\begin{document}

\title{Busulfan with 400 Centigray of Total Body Irradiation and Higher dose
Fludarabine. An alternative regimen for Hematopoietic Stem Cell
Transplantation in Pediatric Acute Lymphoblastic Leukemia.}



\author[1]{Jaspreet Gharial}%
\author[1]{Gregory Guilcher}%
\author[1]{Tony Truong}%
\author[1]{Ravi Shah}%
\author[2]{Sunil Desai}%
\author[2]{Marta Rojas-Vasquez}%
\author[3]{Shahbal Kangarloo}%
\author[1]{Victor Lewis}%
\affil[1]{Alberta Children's Hospital}%
\affil[2]{Stollery Children's Hospital}%
\affil[3]{Tom Baker Cancer Centre}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
\textbf{Background} Hematopoietic stem cell transplantation can be
curative for children with difficult to treat leukemia. The conditioning
regimen utilised is known to influence outcomes. We report outcomes of
the conditioning regimen used at the Alberta Children's Hospital,
consisting of busulfan (with pharmacokinetic target of 3750\selectlanguage{greek}μ\selectlanguage{english}mol*min/day
+/-10\%) for 4 days, higher dose (250 mg/m2) fludarabine and 400
centigray of total body irradiation. \textbf{Procedure} This
retrospective study involved children receiving transplant for acute
lymphoblastic leukemia (ALL). It compared children who fell within the
target range for busulfan with those who were either not measured or
were measured and fell outside this range. All other treatment factors
were identical. \textbf{Results} Twenty-nine children (17 within target)
were evaluated. All subjects engrafted neutrophils with a median (IQR)
time of 14 days (8-30 days). The cumulative incidence of acute graft
versus host disease was 44.8\% (95\% CI 35.6 -- 54.0\%), while chronic
graft versus host disease was noted in 16.0\% (95\% CI 8.7\% - 23.3\%).
At two years, the overall survival was 78.1\% (95\% CI 70.8\% - 86.4\%)
and event free survival was 74.7\% (95\% CI 66.4\% - 83.0\%). Cumulative
incidence of relapse was 11.3\% (95\% CI 5.1\% - 17.5\%). There were no
statistically significant differences in between the group that received
targeted busulfan compared with the untargeted group.
\textbf{Conclusion} The current regimen used in children with ALL
results in outcomes comparable to standard treatment with acceptable
toxicities and significant reduction in radiation dose. Targeting
Busulfan dose in this cohort did not result in improved outcomes.%
\end{abstract}%



\sloppy


\emph{Busulfan with 400 Centigray of Total Body Irradiation and Higher
dose Fludarabine. An alternative regimen for Hematopoietic Stem Cell
Transplantation in Pediatric Acute Lymphoblastic Leukemia.}

\emph{Authors (Academic degrees and affiliations)}

\textbf{Jaspreet Gharial:} MD. Section of Pediatric Hematology Oncology
and Bone Marrow Transplantation, Alberta Children's Hospital. Calgary,
Alberta, Canada.

\textbf{Gregory Guilcher:} BSc, MD, FRCPC, FAAP. Section of Pediatric
Hematology Oncology and Bone Marrow Transplantation, Alberta Children's
Hospital. Calgary, Alberta, Canada.

\textbf{Tony Truong:} MD, MPH, FRCPC. Section of Pediatric Hematology
Oncology and Bone Marrow Transplantation, Alberta Children's Hospital.
Calgary, Alberta, Canada.

\textbf{Ravi Shah:} MD. Section of Pediatric Hematology Oncology and
Bone Marrow Transplantation, Alberta Children's Hospital. Calgary,
Alberta, Canada

\textbf{Sunil Desai:} MBChB, DCH, MRCP(UK), FRCP(C). Division of
Pediatric Hematology/Oncology \& Palliative Care, Stollery Children's
Hospital. Edmonton, Alberta, Canada.

\textbf{Marta Rojas-Vasquez:} MD. Division of Pediatric
Hematology/Oncology \& Palliative Care, Stollery Children's Hospital.
Edmonton, Alberta, Canada.

\textbf{Bill Kangarloo:} Pharmacokinetic Scientist, Alberta Blood and
Marrow Transplant Program, Foothills Hospital, and Tom Baker Cancer
Centre, Calgary, Alberta, Canada

\textbf{Victor Lewis:} MD. Section of Pediatric Hematology Oncology and
Bone Marrow Transplantation, Alberta Children's Hospital. Calgary,
Alberta, Canada.

\emph{Correspondence}

Jaspreet Gharial. 2888 Shaganappi Trail NW, Calgary, Alberta, Canada.
T3B 6A8; Fax +1 (403) 955-2645; Telephone +1 (403) 955-2673; Email:
Jaspreet.gharial@ahs.ca



\emph{Word count}

\textbf{Structured Abstract} : 249 words

\textbf{Main text} (excluding title page, abstract, Conflicts of
Interest, Acknowledgments, References, Tables, Figures, and Legends):
3475 words

\emph{Count of Figures and Tables}

\textbf{Figures:} 5

\textbf{Tables:} 1

\emph{Short Running Title}

Bu, Flu and 400cGy TBI for HSCT in Pediatric ALL.

\emph{Keywords}

Pediatric, Acute Lymphoblastic Leukemia, allogeneic transplant, low-dose
radiation, busulfan, fludarabine



\emph{List of Abbreviations (in alphabetical order)}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
ACH & Alberta Children's Hospital\tabularnewline
\midrule
\endhead
aGvHD & Acute graft versus host disease\tabularnewline
ALL & Acute lymphoblastic leukemia\tabularnewline
ATG & Anti-thymocyte globulin\tabularnewline
AUC & Area under the curve\tabularnewline
BM & Bone marrow\tabularnewline
Bu & Busulfan\tabularnewline
CAR-T & Chimeric antigen receptor T-cell\tabularnewline
cGvHD & Chronic graft versus host disease\tabularnewline
CHC & Chemotherapy-based conditioning\tabularnewline
CI & Confidence interval\tabularnewline
CIR & Cumulative incidences of relapse\tabularnewline
CMV & Cytomegalovirus\tabularnewline
Cy & Cyclophosphamide\tabularnewline
EBV & Epstein Barr Virus\tabularnewline
EFS & Event free survival\tabularnewline
Flu & Fludarabine\tabularnewline
FORUM & For Omitting Radiation Under Majority age\tabularnewline
GvHD & Graft versus host disease\tabularnewline
HLA & Human leukocyte antigen\tabularnewline
HSCT & Hematopoietic stem cell transplantation\tabularnewline
IgG & Immunoglobulin G\tabularnewline
IQR & Interquartile range\tabularnewline
IV & Intravenous\tabularnewline
MRD & Minimal Residual Disease\tabularnewline
NRM & Non-relapse mortality\tabularnewline
OS & Overall survival\tabularnewline
PBSC & Peripheral blood stem cell\tabularnewline
SOS & Sinusoidal obstructive syndrome\tabularnewline
TBI & Total body irradiation\tabularnewline
TDM & Targeted drug monitoring\tabularnewline
Treo & Treosulfan\tabularnewline
\bottomrule
\end{longtable}

\emph{Abstract}

\textbf{Background}

Hematopoietic stem cell transplantation can be curative for children
with difficult to treat leukemia. The conditioning regimen utilised is
known to influence outcomes. We report outcomes of the conditioning
regimen used at the Alberta Children's Hospital, consisting of busulfan
(with pharmacokinetic target of 3750\selectlanguage{greek}μ\selectlanguage{english}mol*min/day +/-10\%) for 4 days,
higher dose (250 mg/m2) fludarabine and 400 centigray of total body
irradiation.

\textbf{Procedure}

This retrospective study involved children receiving transplant for
acute lymphoblastic leukemia (ALL). It compared children who fell within
the target range for busulfan with those who were either not measured or
were measured and fell outside this range. All other treatment factors
were identical.

\textbf{Results}

Twenty-nine children (17 within target) were evaluated. All subjects
engrafted neutrophils with a median (IQR) time of 14 days (8-30 days).
The cumulative incidence of acute graft versus host disease was 44.8\%
(95\% CI 35.6 -- 54.0\%), while chronic graft versus host disease was
noted in 16.0\% (95\% CI 8.7\% - 23.3\%). At two years, the overall
survival was 78.1\% (95\% CI 70.8\% - 86.4\%) and event free survival
was 74.7\% (95\% CI 66.4\% - 83.0\%). Cumulative incidence of relapse
was 11.3\% (95\% CI 5.1\% - 17.5\%). There were no statistically
significant differences in between the group that received targeted
busulfan compared with the untargeted group.

\textbf{Conclusion}

The current regimen used in children with ALL results in outcomes
comparable to standard treatment with acceptable toxicities and
significant reduction in radiation dose. Targeting Busulfan dose in this
cohort did not result in improved outcomes.

\emph{}

\emph{Introduction}

Treatment for acute lymphoblastic leukemia (ALL) is based on established
risk stratification criteria that also include response to therapy. Such
measures have increased the rates of survival from less than 10\% in the
1960s to over 90\% today.\textsuperscript{1}

Hematopoietic stem cell transplantation (HSCT) has a role in the
management of difficult-to-treat leukemia by inducing cures in these
children.\textsuperscript{2,3}. One factor influencing outcomes is the
choice of the conditioning regimen.\textsuperscript{2,4} HSCT regimens
have utilised a combination of total body irradiation (TBI) and high
dose cyclophosphamide (Cy) and/or other agents such as
etoposide.\textsuperscript{5,6,19} However TBI has been associated with
significant adverse effects including neurocognitive decline, endocrine
and metabolic concerns as well as apprehensions in regards to secondary
malignancies.\textsuperscript{7,19} Busulfan (Bu) is a bifunctional DNA
alkylating agent that has been used to replace TBI. Initial studies that
incorporated oral Bu in combination with Cy resulted in poor outcomes
when compared with Cy/TBI.\textsuperscript{7} This was mainly attributed
to the toxicity of oral Bu, specifically the incidence of sinusoidal
obstructive syndrome (SOS).\textsuperscript{5,8} The erratic absorption
of Bu following oral administration with resultant unpredictable
exposure was another contributing factor. This was especially concerning
in pediatric patients owing to their higher drug clearance and less
predictable pharmacokinetic profiles. \textsuperscript{9-11}

The advent of an intravenous formulation of Bu solved the problem of
erratic oral absorption however, interpatient variability in clearance
was a persisting concern. Targeted drug monitoring (TDM) of Bu in
children, once introduced, allowed for better prediction of serum drug
levels.\textsuperscript{49} Although not consistently, subsequent
studies have shown improvements in outcomes following TDM of
Bu.\textsuperscript{12}

At the Alberta Children's Hospital, Bu (with comparatively lower
pharmacokinetic target of 3750\selectlanguage{greek}μ\selectlanguage{english}mol*min/day for 4 days, with a preceding
test dose) is combined with higher dose (250 mg/m\textsuperscript{2})
fludarabine (Flu), 400 cGy of TBI and anti-thymocyte globulin (ATG) in a
regimen that was initially used in adults and later adapted for HSCT in
children.\textsuperscript{13}

We report our experience with this conditioning regimen for children
diagnosed with ALL requiring HSCT.

\emph{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
3750\selectlanguage{greek}μ\selectlanguage{english}mol*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/m\textsuperscript{2} infused IV over one hour once
. Rabbit - ATG (Thymoglobulin \selectlanguage{ngerman}® 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/m\textsuperscript{2} was
administered on day 1 and 10mg/m\textsuperscript{2} 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).\textsuperscript{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/\selectlanguage{greek}μ\selectlanguage{english}l
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.

\emph{Results}

Patient Demographics

Over the 18-year period from 2003 to 2020, 29 patients met eligibility
criteria. They were split into the targeted group which comprised of 17
subjects and the untargeted group which was made up of the remaining 12
subjects.

Patient Demographic data is presented in Table 1. The recipients had a
median age of 13 years (range 2-18 years) and 72\% were male. All
patients were in morphologic remission at the time of transplant.
Fourteen patients (48\%) were in CR1 and were transplanted due to
high-risk disease: Eight had persistently positive Minimal Residual
Disease (MRD), three were Philadelphia or Philadelphia like chromosome
positive, two had hypodiploid ALL and one patient had bi-phenotypic
leukemia. The remaining 15 (52\%) were transplanted in CR2. Data on MRD
status prior to transplant was available for 25 subjects. Three-quarters
of this group (65\% of the entire cohort) had a level below 0.01\% by
flow cytometric analysis and were considered negative.

Engraftment

Neutrophil engraftment was seen in all patients at a median of 14 days
(range: 8-30 days). Platelet engraftment was successful in 28 of the 29
patients, the remaining one died prior to engraftment. The median time
to platelet engraftment was 16 days (range 0-89 days). Two patients had
engraftment beyond day 60, one of whom required continued platelet
infusion due to persistent hematuria.

Graft versus host disease

Fourteen of the 29 subjects developed grade 2 or higher aGvHD, resulting
in a cumulative incidence at day 100 of 44.8\% (95\% CI 35.6 -- 54.0\%).
Severe aGVHD (grade three or higher) was seen in six patients which gave
a cumulative incidence at day 100 of 20.7\% (95\% CI 9.9 -- 40.3\%).

Chronic graft versus host disease (cGvHD) was seen in four subjects. The
cumulative incidence of cGvHD at one year was 16.0\% (95\% CI 8.7\% -
23.3\%). cGvHD was graded as severe in three of the four subjects. All
three had pre-existing aGvHD.

Toxicity

CMV infection was documented in nine of 29 subjects in the cohort giving
a cumulative incidence of 31\% (95\% CI 22.4\% - 39.6\%) at one year.
The median time to reactivation was 24 days (IQR of 16 - 34 days). Two
of these 9 children had a primary infection. Clinically significant EBV
reactivations that required pre-emptive treatment were documented in
five of 27 patients for whom data was available. Culture proven
bacterial infections were documented in nine subjects within the first
100 days post-transplant. Gram-positive organisms were cultured in blood
in two-thirds of positive cases. Finally fungal infections were seen in
two subjects, both of whom developed oral thrush.

Grade three mucositis was seen in all 29 subjects in this analysis.
Sinusoidal obstructive syndrome (SOS) was seen in three of 20 patients
in the cohort for whom data was available. The cumulative incidence at
100 days post transplant was 15.0\% (95\% CI 7.0\% - 23.0\%). Only one
participant had severe SOS resulting in death from multi-organ
dysfunction.

Survival analysis

The median follow-up for the cohort was 3.25 years (1-10 years). There
were six deaths recorded, all occurring within two years of transplant
resulting in a 2-year overall survival for the entire cohort of 78.1\%
(95\% CI 70.8\% - 86.4\%). The Bu targeted group had a similar 2-year
overall survival calculated at 79.9\% (95\% CI 69.4\% - 90.4\%) compared
to the untargeted group which was 75.0\% (95\% CI 62.5\% - 87.5\%). This
difference did not reach statistical significance, p value = 0.6 (Figure
2).

In the subjects who succumbed, two deaths were due to progressive
disease post relapse, one patient was lost to SOS and another to causes
directly related to cGvHD. The remaining two children died due to
progressive organ dysfunction.

Overall, there were a total of seven events. No events occurred after 2
years of transplantation. This resulted in a 2-year EFS of 74.7\% (95\%
CI 66.4\% -- 83.0\%). In the Bu targeted group, the 2-year EFS was
79.9\% (95\% CI 69.4\% - 90.4\%) compared to the untargeted group:
66.7\% (95\% CI 53.1\% - 80.3\%), p = 0.4. (Figure 3).

Three children in the cohort relapsed post-transplant, giving a
cumulative incidence of relapse at two years of 11.3\% (95\% CI 5.1\% -
17.5\%). One of the three children went on to receive a successful
second transplant (Figure 4).

The non-relapse mortality calculated at two years was 15.4\% (95\% CI
8.3\% - 22.5\%) for the entire group and was 15.4\% (95\% CI 5.4\% -
25.4\%) and 16.7\% (95\% CI 5.9\% - 27.5\%) p = 0.7 in the Bu targeted
and untargeted groups respectively (Figure 5).

Univariate analysis did not reveal any factors, including pre-transplant
MRD status, that correlated significantly with the risk of death or
relapse.

\emph{Discussion}

The conditioning regimen used for our children with acute leukemia was
initially developed for adult transplant recipients. It included Bu
(targeted at an AUC of 3750\selectlanguage{greek}μ\selectlanguage{english}mol*min) and Flu (dosed at
250mg/m\textsuperscript{2} total), to which ATG was added to reduce the
incidence of GvHD.\textsuperscript{13,16} TBI at 400cGy was included in
the protocol to reduce the incidence of relapse and was noted to
significantly improve outcomes.\textsuperscript{17} Our protocol uses a
higher Flu dose compared with other centers using similar regimens.

Survival outcomes, relapse incidence and non-relapse mortality in our
cohort were similar between the subjects who had TDM and those who did
not. The number of subjects in our cohort played an important role in
the results not reaching significance levels. Previous studies that have
compared Bu based conditioning to TBI regimens in children with
malignancies have presented outcomes favouring TBI based
conditioning.\textsuperscript{8,18} This has mainly been due to higher
rates of relapse and non-relapse mortality rates in the Bu groups.
However, these earlier studies have not consistently reported
measurements of Bu serum levels.\textsuperscript{8,18,47}

Recently, the results of the international FORUM study (For Omitting
Radiation Under Majority age), a multicenter randomised trial comparing
a fractionated TBI/Etoposide regimen to various chemotherapy-based
conditioning (CHC) regimens has shown conclusively that a TBI based
conditioning regimen is superior for children above four years of age
with ALL.\textsuperscript{19} In that study individual institutions were
allowed to choose between Bu and Treosulfan (Treo) as a backbone for
their CHC regimens. A potential drawback to this flexibility is the
introduction of heterogeneity.\textsuperscript{51} Therefore, despite an
overall equivalence between the Bu and Treo based regimens, data does
not allow a direct comparison across individual CHCs. Furthermore, Bu
serum level targeting was allowed but not mandated by the FORUM study
and no analysis is done to compare outcomes between the centers that
used serum targeting of Bu and those that did
not.\textsuperscript{51}Additionally, although cranial radiation was
allowed for central nervous system positive disease, none of the CHC
regimens in the FORUM study included TBI. For these reasons, a direct
comparison of outcomes with the regimen used in the current study is not
appropriate.

In 2007, Chaudhury et al published an abstract reporting their findings
using a regimen incorporating both Bu and
TBI.\textsuperscript{20}Although the targets for Bu TDM were similar
(4000 \selectlanguage{greek}μ\selectlanguage{english}mol *min/day), an Important difference in their preparation
regimen was their use of lower cumulative doses of Flu (150
mg/m\textsuperscript{2} over five days). They reported a one-year OS and
PFS of 63\% and 56\% respectively. A recent article by Rosoff et al
further expanding analysis of the same cohort, using Bu-Flu-ATG and
400cGy TBI regimen, reported a five-year OS of 37\% in their ALL cohort
consisting of only nine children (one of whom had positive
MRD).\textsuperscript{21} In both studies by Chaudhury and Rosoff the
most important reason for failure was high rates of relapse, seen in
29\% and 28\% of subjects respectively.\textsuperscript{20,21} We
speculate that the lower cumulative incidence of relapse in our cohort
may potentially be related to the higher doses of Flu. Evidence for this
has recently been published in both transplant and Chimeric Antigen
Receptor T-cell (CAR-T) settings with studies showing that both lower
and very high serum Flu levels result in inferior outcomes driven by
increased relapse rates and toxicity
respectively.\textsuperscript{52,53}

Although the rates of aGvHD reported in this cohort are higher than
those in other studies, which quote incidences closer to
30\%,\textsuperscript{18,21,23} they are similar to results reported by
Chaudhury et al who report their severe aGvHD rate as being
32\%.\textsuperscript{20} A striking similarity between both studies is
the use of PBSC products, utilised in close to 90\% of transplants in
the current study and in 75\% of cases in the study by Chaudhury. PBSC
products have been shown to be associated with increased aGvHD in
multiple studies although the difference has not been consistently
significant.\textsuperscript{24,25,48} Six (20\%) of the donor-recipient
pairs in our study were female donors to male recipients. Pulsipher et
al, have shown that the presence of aGvHD is related to a lower risk of
relapse,\textsuperscript{50} which perhaps contributed to the EFS in the
current study.

The cumulative incidence of cGvHD in our study was comparable with most
other studies. \textsuperscript{18,20,29} It was lower than rates
reported by Rosoff et al and those in a report by Modi et al (which used
a similar conditioning regimen for adults with
AML).\textsuperscript{21,23} The reason for the low rates of cGvHD in
our study despite a higher incidence of aGvHD may be explained by our
method of giving ATG.\textsuperscript{30,31} At our institution we
follow a regimen similar to one recommended by Bacigalupo et
al.\textsuperscript{31} In their 2001 paper, they suggested all patients
receive ATG at 7.5mg/kg as this dose was not associated with increased
risk of infections and was shown to reduce the incidence of cGvHD.

The incidence and timing of viral infections seen in our cohort were
similar to those reported in literature\textsuperscript{32-34,36} with
the exception of CMV which appeared to occur earlier than some studies
have described (median of 24 days in our study compared with 33 to 41
days in some studies).\textsuperscript{34,54} There was no effect on
outcomes which is contrary to findings in other
publications,\textsuperscript{32,35} with the exception of delayed
platelet engraftment dates in those subjects receiving platelet
infusions for hematuria secondary to BK virus induced cystitis Bacterial
blood stream infection incidence, timing and the organisms grown were
also comparable to other reports.\textsuperscript{37-41} The incidence
of mucositis was 100\% in this cohort which is in keeping with other
reports using a similar regimen.\textsuperscript{21}

The incidence of SOS in studies using conditioning regimens including Bu
and Flu compared to other combinations have had mixed results with some
studies reporting a reduction in SOS\textsuperscript{42-44} and others
showing no difference.\textsuperscript{45} In our cohort the cumulative
incidence of SOS at d100 was 15\% (three patients). Although this rate
appears higher compared with other studies, it may not be significant
owing to the small number of patients developing
SOS.\textsuperscript{20,46} In the adult study by Kabriaei et al, six
patients in their cohort of 107 patients developed SOS resulting in a
prevalence of 6\%.\textsuperscript{46} Similarly, Chaudhury et al
reported a SOS prevalence of 10\% (three cases).\textsuperscript{6} It
is notable that SOS was cited as a direct cause of death in only one
case in each of the two studies. The univariate analysis in our cohort
revealed no significant correlation of the occurrence of SOS with
mortality.

The limitations to our study include firstly, that this is a
retrospective chart review and therefore dependant on the quality of
data collected and stored. Data pertaining to SOS were missing for our
earlier subjects. Secondly, the number of patients, once split into the
two cohorts, was limited and this may have contributed to the lack of
significance when comparing the outcomes following Bu pharmacokinetic
targeting. Furthermore, the comparison with a historic cohort introduces
some inconsistencies as there is a possibility that improvements in
outcomes over the years were in fact due to an unidentified confounder
and not the practice of targeting Bu serum levels. It should be noted
however, that no significant changes to supportive care were introduced
during the period of data collection. Related to this, the combination
during analysis of those children who did not have any Bu monitoring
with the group that were monitored and were out of range may have
introduced a bias, as the earlier group is a heterogenous one and may
contain some children who were within acceptable serum levels. Finally,
we did not differentiate between T-cell and B-cell ALL which introduces
heterogeneity in the cohort analysed. Despite these limitations, the
paper documents outcomes following transplant in children with ALL using
a unique and previously unpublished regimen of Bu-ATG and lower dose TBI
regimen combined with a higher dose of Flu. Although previous studies
have utilized lower dose radiation in this setting, those cohorts have
been of limited size compared with the current report. We have
demonstrated similar outcomes to other conditioning regimens when serum
Bu levels are targeted suggesting this may be a substitute to consider
when lowering radiation dose is desirable.

\emph{Conclusion}

The use of a conditioning regimen for allogeneic hematopoietic cell
transplantation in children with ALL using Bu along with high dose Flu,
ATG and 400 Centigray of TBI results in outcomes comparable to standard
treatment with acceptable toxicities and significant reduction in the
dose of irradiation. Targeting the Bu dose based on pharmacokinetic
monitoring did not however improve outcomes in this cohort.

\emph{Conflict of Interest Statement}

The authors have no conflicts of interest to declare.

\emph{Acknowledgements}

The authors would like to acknowledge the contribution of the Alberta
Health Services.

\emph{References}

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\emph{Figure Legends:}

\textbf{Figure 1:} graphical representation of the cohorts for analysis

\textbf{Figure 2:} Overall survival stratified for busulfan
pharmacokinetic targeting. The targeted group 3-year OS of 79.9\% (95\%
CI 69.4\% - 90.4\%) compared with the untargeted group: 75.0\% (95\% CI
62.5\% - 87.5\%). P value = 0.6

\textbf{Figure 3:} Event Free survival stratified for busulfan
pharmacokinetic targeting. The targeted group 2-year EFS of 79.9\% (95\%
CI 69.4\% - 90.4\%) compared with the untargeted group: 66.7\% (95\% CI
53.1\% - 80.3\%). P value = 0.4

\textbf{Figure 4:} Cumulative incidence of relapse (CIR). 3 events, with
CIR = 11.3\% (95\% CI 5.1\% - 17.5\%)

\textbf{Figure 5:} Non-Relapse mortality stratified for busulfan
pharmacokinetic levels. The targeted group NRM of 15.4\% (95\% CI 5.4\%
- 25.4\%) compared with the untargeted group: 16.7\% (95\% CI 5.9\% -
27.5\%). P value = 0.7\selectlanguage{english}
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