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\begin{document}

\title{Targeted busulfan-based conditioning unrelated donor bone marrow
transplantation for Diamond-Blackfan anemia}



\author[1]{Katsuhide Eguchi}%
\author[1]{Masataka Ishimura}%
\author[2]{Akihiro Tamura}%
\author[3]{Nobuhiro Ito}%
\author[4]{Naoki Hirano}%
\author[1]{Shunichi Adachi}%
\author[1]{Keishiro Kinoshita}%
\author[1]{Hirokazu Kobushi}%
\author[1]{Yutaro Yada}%
\author[1]{Motoshi Sonoda}%
\author[1]{Akira Shiraishi}%
\author[2]{Yoshiyuki Kosaka}%
\author[5]{Kiminori Terui}%
\author[5]{Etsuro Ito}%
\author[1]{Shouichi Ohga}%
\affil[1]{Graduate School of Medicial Sciences, Kyushu University}%
\affil[2]{Kobe Children’s Hospital}%
\affil[3]{Nagasaki University Hospital}%
\affil[4]{Oita University Faculty of Medicine}%
\affil[5]{Hirosaki University Graduate School of Medicine}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Hematopoietic cell transplantation corrects a hematological phenotype of
Diamond-Blackfan anemia (DBA). Reduced conditioning has been recommended
for patients with the cancer predisposition, but appropriate intensity
is required to prevent graft failure. We conducted unrelated-donor bone
marrow transplantations in six consecutive transfusion-dependent
patients (median age, 5.4 years; range, 1.8--23) after targeted-busulfan
(60--75 mg/L\selectlanguage{ngerman}×h of cumulative AUC) and fludarabine regimen without
irradiation. All obtained chronic graft-versus-host-disease-free
complete donor chimerism, including one unrelated cord blood
transplantation rescue. The rescued 7-year-old girl had the longest
transfusion-dependency (\textgreater{}40 months) and highest ferritin
level (\textgreater{}2,000 ng/mL) pretransplant. Intermediate-intensity
targeted busulfan-based conditioning may cure DBA.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Targeted busulfan-based conditioning unrelated donor bone marrow
transplantation for Diamond-Blackfan anemia}

Katsuhide Eguchi\textsuperscript{1)}, Masataka
Ishimura\textsuperscript{1)}, Akihiro Tamura\textsuperscript{2)},
Nobuhiro Ito\textsuperscript{3)}, Naoki Hirano\textsuperscript{4)},
Shunichi Adachi\textsuperscript{1)}, Keishiro
Kinoshita\textsuperscript{1)}, Hirokazu Kobushi\textsuperscript{1)},
Yutaro Yada\textsuperscript{1)}, Motoshi Sonoda\textsuperscript{1)},
Akira Shiraishi\textsuperscript{1)}, Yoshiyuki
Kosaka\textsuperscript{2)}, Kiminori Terui\textsuperscript{5)}, Etsuro
Ito\textsuperscript{5)}, Shouichi Ohga\textsuperscript{1)}

\textbf{Affiliations} : 1. Department of Pediatrics, Graduate School of
Medical Sciences, Kyushu University, Fukuoka, Japan, 2. Department of
Hematology and Oncology, Kobe Children's Hospital, Hyogo, Japan, 3.
Department of Pediatrics, Nagasaki University Hospital, Nagasaki, Japan,
4. Department of Pediatrics, Oita University Faculty of Medicine, Oita,
Japan, 5. Department of Pediatrics, Hirosaki University Graduate School
of Medicine, Aomori, Japan.

\textbf{Corresponding author} : Masataka Ishimura, Department of
Pediatrics, Graduate School of Medical Sciences, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. PHONE:
+81-92-642-5421, FAX: +81-92-642-5435, E-mail:
ischii@pediatr.med.kyushu-u.ac.jp

\begin{CJK}{UTF8}{gbsn}\textbf{Word count　} \end{CJK}\selectlanguage{english}unstructured abstract: 100 words
(\emph{\textless{}} 100), text: 1191 words (\emph{\textless{}}
1200\textbf{)}

\begin{CJK}{UTF8}{gbsn}\textbf{Number of tables and figures　} \end{CJK}\selectlanguage{english}TABLE: 1, Figure: 1 (up to 2),
References: 20 (as needed)

\textbf{Short running title:} Targeted BU-conditioning unrelated BMT for
DBA

\textbf{Keywords:} Diamond-Blackfan anemia; targeted busulfan;
post-transplant neoplasms

\textbf{Abbreviations:}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
AUC & Area-under-the-curve\tabularnewline
\midrule
\endhead
BMT & Bone marrow transplantation\tabularnewline
BU & Busulfan\tabularnewline
CB & Cord blood\tabularnewline
DBA & Diamond-Blackfan anemia\tabularnewline
EBMT & European Blood and Marrow Transplantation Group\tabularnewline
ESID & European Society for Immunodeficiencies\tabularnewline
FLU & Fludarabine\tabularnewline
GVHD & Graft-versus-host disease\tabularnewline
HCT & Hematopoietic cell transplantation\tabularnewline
IBMFS & Inherited bone marrow failure syndrome\tabularnewline
MAC & Myeloablative conditioning\tabularnewline
MEL & Melphalan\tabularnewline
PRCA & Pure red cell aplasia\tabularnewline
rATG & Rabbit anti-thymocyte globulin\tabularnewline
RIC & Reduced intensity conditioning\tabularnewline
RP & Ribosomal protein\tabularnewline
RRT & Regimen related toxicities\tabularnewline
TBI & Total body irradiation\tabularnewline
Treo & Treosulfan\tabularnewline
\bottomrule
\end{longtable}



\textbf{ABSTRACT}

Hematopoietic cell transplantation corrects a hematological phenotype of
Diamond-Blackfan anemia (DBA). Reduced conditioning has been recommended
for patients with the cancer predisposition, but appropriate intensity
is required to prevent graft failure. We conducted unrelated-donor bone
marrow transplantations in six consecutive transfusion-dependent
patients (median age, 5.4 years; range, 1.8--23) after targeted-busulfan
(60--75 mg/L\selectlanguage{ngerman}×h of cumulative AUC) and fludarabine regimen without
irradiation. All obtained chronic graft-versus-host-disease-free
complete donor chimerism, including one unrelated cord blood
transplantation rescue. The rescued 7-year-old girl had the longest
transfusion-dependency (\textgreater{}40 months) and highest ferritin
level (\textgreater{}2,000 ng/mL) pretransplant. Intermediate-intensity
targeted busulfan-based conditioning may cure DBA.



\textbf{INTRODUCTION}

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure
syndrome (IBMFS) characterized by hypoproliferative and proapoptotic
erythropoiesis along with malformations and cancer
predisposition.\textsuperscript{1} The pure red cell aplasia (PRCA)
arises from haploinsufficiency due to a loss of function mutation in
ribosomal protein (RP)-encoding genes. Corticosteroids are the
first-line treatment for PRCA in infancy, but \textgreater{}20\% of
patients result in transfusion-dependency. Patients are predisposed to
myeloid malignancy and solid tumors. In pediatric patients, the 5-year
chronic graft-versus-host-disease (GVHD)-free survival rate following
allogeneic hematopoietic cell transplantation (HCT) has improved to
87.0\%, including mostly histocompatible sibling or unrelated
donors.\textsuperscript{2} HCT can achieve a hematological cure
transfusion-dependent young patients if a suitable donor is available.
Nevertheless, conditioning of alternate donor HCT is carefully
personalized according to organ dysfunctions associated with the
ribosomopathy by itself and iron-overload. Regarding cancer
predisposition and the high risk of complications, reduced intensity
conditioning (RIC) is preferable; however, there is limited information
on unrelated donor HCT.\textsuperscript{3-7} To optimize the HCT regimen
for DBA, we assessed the outcomes of recent cases with alternate donor
bone marrow transplantation (BMT) following targeted busulfan (BU)-based
intermediate intensity conditioning.

\textbf{METHODS}

Among 7 patients with RP-mutated DBA who received HCT in Kyushu
University Hospital (n=6) and Kobe Children's Hospital (n=1) from 2000
to 2020, the latest six patients received targeted BU and fludarabine
(FLU) conditioning in the first HCT. BU was administered intravenously
four times daily for four days. The cumulative area-under-the-curve
(AUC) of BU was calculated by JMP Pro 11 (version 11.2.0 for Windows;
JMP Inc., SAS Institute Japan) based on the results of a test-dose using
the Dried Blood Spot method. Based on the transfusion-dependent periods
and serum ferritin levels, the target of cumulative AUC was adjusted to
60--75 mg/L×h (65\textasciitilde{}80\% of the conventional myeloablative
cumulative AUC {[}85--95 mg/L×h{]}). FLU was administered at 180
mg/m\textsuperscript{2} in four to six divided doses. Rabbit
anti-thymocyte globulin (rATG) (2.5--5.0 mg/kg
Thymoglobulin\selectlanguage{english}\textsuperscript{}, Genzyme, Boston, MA) was added in five
patients. Tacrolimus and short-term methotrexate were used for acute
GVHD prophylaxis in all patients. The date of engraftment was defined as
the first of three consecutive days when the neutrophil count was
\textgreater{}0.5x10\textsuperscript{9}/L. Donor chimerism was assessed
by sex chromosome fluorescence \emph{in situ} hybridization or short
tandem repeats.

\textbf{RESULTS}

\textbf{Pretransplant State of the Case-Series}

From October 2015 to March 2021, 6 patients with DBA consecutively
received unrelated-donor BMT due to steroid-resistant and
transfusion-dependent anemia. The detailed clinical profiles are shown
in \textbf{TABLE 1} . Mutations were identified in \emph{RPS19} in 5
patients and \emph{RPS26} in 1 patient. The median age at the time of
BMT was 5.4 years (range, 1.8--23 years). All patients received
unrelated-donor bone marrow (HLA-full allele-matched donor, n=3; HLA-one
locus-mismatched donor, n=3). The median transfusion-dependent period
was 31 months (range, 4--78 months) and the median pre-transplant serum
ferritin level was 1164.4 ng/mL (range, 271.7--2174.0 ng/mL). The median
cumulative AUC of BU was 61 mg/Lxh (range, 60--75 mg/Lxh), and the
median dose per body weight was 12.0 mg/kg (range, 8.4--14.6 mg/kg). One
patient had medication-free atrial septal defect at potential risk of
transplantation. The other ribosomopathy-associated defects were not
considered a risk of this conditioning. Three had over ferritinemia
(\textgreater{}1000 ng/mL) but no evidence of organ dysfunction with
hemosiderosis.

\textbf{Post-transplant Outcomes}

Five patients achieved neutrophil engraftment at the median of 17 days
(range, 13--21 days), and all had sustained \textgreater{}95\% donor
chimerism without donor lymphocyte infusion. One (Patient 4) developed
sinusoidal obstruction syndrome and a graft failure despite well
targeted BU. She had the longest transfusion-dependent period and the
highest ferritin level of all patients (\textbf{Fig. 1} ). Two months
later, umbilical cord blood (CB) transplantation was successfully
performed after our conventional regimen FLU, melphalan (MEL) and
low-dose total body irradiation (TBI). Histocompatible CB was obtained
from the Japanese CB Bank Network. She is presently active in her school
life with complete donor chimerism.

No grade \textgreater{}II acute GVHD or chronic GVHD occurred. Acute
GVHD was well controlled by corticosteroids. Infectious complications
occurred and were controlled in all 3 patients, including
cytomegalovirus reactivation (n=2), human herpes virus-6 reactivation
(n=1), non-tuberculosis mycobacteria infection (n=1), and herpes zoster
(n=1). Autoimmune cytopenia occurred on treatment in one patient after
complete engraftment. During the median follow-up period after HCT (42.5
months {[}range, 6--71 months{]}), no patients had neurological
disability, neoplasms or pulmonary complications.

\textbf{DISCUSSION}

Five transfusion-dependent DBA patients successfully underwent targeted
BU-based conditioning 3 HLA-matched or 2 one-locus HLA-mismatched
unrelated donor BMT; in the latter 2 cases, transplantation was at
\textgreater{}10 and \textgreater{}20 years of age. The longest
transfusion-dependent case, which showed the highest ferritinemia had
one graft failure but was rescued by CB transplantation after our
established low-dose TBI regimen. The targeted BU-based intermediate
intensity regimen was optimal for alternate donor BMT in DBA patients.

For successful HCT, the diverse clinical expression and high penetrance
of DBA hamper the search for suitable family donors. Organ dysfunction
associated with anomaly and/or iron overload increases the risk of
regimen related toxicities (RRTs). Repeated transfusion augments the
risk of rejection. RIC is preferable to reduce RRT and late
complications in IBMFS. In a recent report from
Brazil,\textsuperscript{8} the 5-year overall survival rates after
HLA-matched sibling donor, HLA-matched unrelated donor, and
HLA-mismatched donor HCT were 80\%, 73\%, and 29\%, respectively. In the
EBMT database, the 3-year event-free survival rate of patients who
received unrelated donor HCT was
\textgreater{}80\%.\textsuperscript{9}In Japan,\textsuperscript{3} 8 of
9 DBA patients underwent successful RIC-unrelated donor BMT, but all 9
patients received RIC regimen with both irradiation and alkylators.
Appreciable intensity is required for complete donor chimerism to reduce
the risk of graft failure and myeloid malignancy. TBI should be avoided
in conditioning regimens for non-malignant disease, especially for
cancer predisposition syndromes. BU or treosulfan (Treo) and FLU-based
myeloablative conditioning (MAC) has been recommended for HCT for DBA
patients.\textsuperscript{10} Treo and alemtuzumab-RIC has been reported
in a limited number of cases;\textsuperscript{4-7} however, neither is
licensed in Japan. Our targeted BU-based regimen was effectively used
for children and adults. The dosage can be further personalized for
wider ranges of circulating BU levels in infants.\textsuperscript{11}
Alemtuzumab is more effective than rATG in preventing GVHD in patients
with non-malignant disorders.\textsuperscript{12} Replacement of rATG
with alemtuzumab may improve engraftment and decrease chronic GVHD
without resulting in delayed immune reconstitution.\textsuperscript{13}
Because chronic GVHD increases the risk of post-transplant neoplasms in
DBA patients,\textsuperscript{14} rATG would be replaced by alemtuzumab.
More recently, a critical precaution of Treo dose individualization has
been reported in infants and children undergoing allo-HCT for
non-malignant conditions.\textsuperscript{15} Precise targeted
conditioning is needed for pediatric patients even in the setting of BU
or Treo.

Colorectal cancer, osteogenic sarcoma and cardiac Purkinje cell tumor
have been reported in DBA patients, most frequently at a median of 9
years after MAC-HCT.\textsuperscript{16-19} The combination of
alkylators and TBI raises the risk of osteogenic sarcoma in pediatric
patients after MAC-HCT;\textsuperscript{20} however, there is no
information on the effect of a single alkylator in RIC-HCT. The cancer
risk and clonal hematopoiesis may be more accelerated with age in IBMFS.
Cancer vigilance is needed to optimize the time and conditioning of DBA
patients as well as the age at HCT.

\textbf{Acknowledgements:} We thank to all of the staff who participated
in the treatment of patients in Kyushu University (Fukuoka, Japan) and
Kobe Children's Hospital (Hyogo, Japan), and the staff members of
Hirosaki University (Aomori, Japan) for the genetic analysis of the
patient. This work was supported by the Health and Labor Sciences
Research grants under Grant Number 19FC1005.

\textbf{Conflict of Interest Statement:} The authors declare no conflict
of interest in association with the present study.

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\textbf{Figure legend}

\textbf{Figure 1.} Relationship among the cumulative AUC of BU, the
transfusion-dependent period, and ferritin levels.

Each number represents the patient number in Table 1. The radii of
circles show the pre-transplant serum ferritin levels in individuals.
White and black circles represent engraftment and rejection after the
first HCT, respectively. Thin and thick shaded areas indicate the RIC
and MAC range, respectively, in the EBMT/ESID guideline defined by the
cumulative AUC of BU.

\textbf{Hosted file}

\verb`Table-PBC DBA SCT.docx`  available at \url{https://authorea.com/users/737600/articles/712457-targeted-busulfan-based-conditioning-unrelated-donor-bone-marrow-transplantation-for-diamond-blackfan-anemia}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-PBC-DBA-SCT-submit/Figure-PBC-DBA-SCT-submit}

\end{center}
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\selectlanguage{english}
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\end{document}

