Transplantation and Cellular Therapy 30 (2024) 105.e1
105.

e10

Transplantation and
Cellular Therapy
journal homepage: www.astctjournal.org

Full Length Article

Pediatric

Allogeneic Hematopoietic Cell Transplantation for Juvenile

Myelomonocytic Leukemia with a Busulfan, Fludarabine, and
Melphalan Regimen: JPLSG JMML-11
Kazuo Sakashita1,*, Nao Yoshida2, Hideki Muramatsu3, Yoshitoshi Ohtsuka4,
Kenichiro Watanabe5, Miharu Yabe6, Harumi Kakuda7, Yuko Honda8,
Tomoyuki Watanabe9, Masami Haba10, Shigeru Ohmori11, Kazuyuki Matsuda12,
Yuki Yuza13, Akiko Saito14, Keizo Horibe14, Souichi Adachi15, Atsushi Manabe16
1
Department of Hematology and Oncology, Nagano Children’s Hospital, Azumino, Japan
2
Department of Hematology and Oncology, Children’s Medical Center, Japanese Red Cross Aichi Medical Center Nagoya First
Hospital, Nagoya, Japan
3
Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan
4
Department of Pediatrics, Hyogo College of Medicine, Nishinomiya, Japan
5
Department of Hematology and Oncology, Shizuoka Children’s Hospital, Sizuoka, Japan
6
Department of Innovative Medical Science, Tokai University School of Medicine, Isehara, Japan
7
Department of Hematology and Oncology, Chiba Children’s Hospital, Chiba, Japan
8
Department of Pediatrics, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
9
Department of Health and Nutritional Sciences, Faculty of Health Sciences, Aichi Gakuin University, Nisshin, Japan
10
Department of Biopharmaceutics, Faculty of Pharmacy, Chiba Institute of Science, Choshi, Japan
11
Department of Pharmacy, Shinshu University Hospital, Matsumoto, Japan
12
Department of Clinical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan
13
Department of Pediatric Hematology Oncology, Tokyo Metropolitan Children’s Medical Center, Fuchu, Japan
14
Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
15
Human Health Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan
16
Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Article history: A B S T R A C T
Received 20 June 2023 Juvenile myelomonocytic leukemia (JMML), which is classified as a myelodysplastic/mye-
Accepted 2 October 2023 loproliferative neoplasm, is a rare hematologic malignancy of childhood. Most patients
with JMML require allogeneic hematopoietic cell transplantation (HCT) as a curative ther-
Key words:
apy. A Japanese retrospective analysis demonstrated favorable outcomes for a busulfan
Juvenile myelomono-
(BU) + fludarabine (FLU) + melphalan (MEL) regimen, with an overall survival (OS) of 72%
cytic leukemia
Hematopoietic cell and an event-free survival (EFS) of 53%. To further validate the efficacy and safety of this
transplantation regimen, the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) conducted a
Busulfan nationwide prospective study, JMML-11. Between July 2011 and June 2017, 28 patients
Fludarabine with newly diagnosed JMML were enrolled in JMML11. Low-dose chemotherapy for
Melphalan tumor control before HCT was recommended, and patients treated with AML-type

Financial disclosure: See Acknowledgments on page 105.e9.

*Correspondence and reprint requests: Kazuo Sakashita, MD, PhD, Department of Hematology and Oncology, Nagano Child-
ren’s Hospital, 3100 Toyoshina, Azumino, Nagano 399-8205, Japan
E-mail address: [email protected] (K. Sakashita).

https://doi.org/10.1016/j.jtct.2023.10.002
2666-6367/© 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights
reserved.
105.e2 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10

chemotherapy and azacitidine were excluded. The conditioning regimen comprised i.v.
BU, 16 doses administered every 6 h, with dose adjustment based on pharmacokinetic
(PK) studies on days -11 to -8; FLU, 30 mg/m2/day or 1 mg/kg/day for patients <10 kg or
age <1 year on days -7 to -4; and MEL, 90 mg/m2/day or 3 mg/kg/day for patients <10 kg
or <1 year on days -3 to -2. The donor was selected by the physician in charge. A family
donor was available for 7 patients (3 HLA-matched siblings, 3 HLA-1-antigen mismatched
parents, and 1 haploidentical father). Overall, 21 patients received grafts from unrelated
donors, including 8 HLA-matched donors and 13 HLA-mismatched donors. The graft
source was related bone marrow (BM) for 7 patients, unrelated BM for 14 patients, and
unrelated cord blood for 7 patients. Neutrophil engraftment was achieved in 21 of 28
patients (75%), with a median of 20.5 days (range, 11 to 39 days) after transplantation.
The 3-year OS, 3-year EFS, 3-year relapse rate, and 3-year transplantation-related mortal-
ity were 63% (95% confidence interval [CI], 42% to 78%), 52% (95% CI, 32% to 69%), 18%
(95% CI, 6% to 34%), and 21% (95% CI, 9% to 38%), respectively. WBC count before the con-
ditioning regimen (
7.0 £ 109/L) was significantly associated with inferior EFS and OS.
Body surface area
.5 m2, spleen size <4 cm before conditioning, and HLA-matched unre-
lated BM donors were significantly associated with better OS. Adverse effects related to
the conditioning regimen included febrile neutropenia (86%), diarrhea (39%), hypoxemia
(21%), and mucositis (18%). BU-associated toxicity, including sinusoidal obstruction syn-
drome (SOS) and thrombotic microangiopathy (TMA), occurred in 7 patients (25%; SOS,
n = 6; TMA, n = 2). Retrospective analysis of PK data after the first BU dose in 23 patients,
including 6 with SOS and 17 without SOS, did not show significant differences between
groups. The JMML-11 study confirms the positive results of previous retrospective analy-
ses. BU+FLU+MEL might become a standard conditioning regimen for patients with
JMML.
© 2023 The American Society for Transplantation and Cellular Therapy. Published by
Elsevier Inc. All rights reserved.

INTRODUCTION free survival (EFS) rates of 60% and 52%, respec-

Juvenile myelomonocytic leukemia (JMML) is a tively. A Japanese retrospective analysis demon-
rare hematologic malignancy of infancy and child- strated favorable results for the BU + fludarabine
hood and is classified as a myelodysplastic/myelo- (FLU) + MEL regimen, with an OS of 72% and an EFS
proliferative neoplasm according to the World of 53% [9]. To validate the efficacy and safety of this
Health Organization [1]. JMML is characterized by regimen, the Japan Pediatric Leukemia/Lymphoma
granulocyte-macrophage colony-stimulating factor Study Group (JPLSG) conducted a nationwide pro-
hypersensitivity and genetic mutations in the RAS/ spective study, JMML-11. Twenty-eight JMML
MAPK signaling pathway [2]. Up to 90% of children patients were enrolled in the study, which showed
with JMML exhibit somatic or germline abnormali- that BU + FLU + MEL may become a standard condi-
ties in PTPN11, NF1, KRAS, NRAS, or CBL [3]. tioning regimen for HCT in patients with JMML.
Patients with JMML are treated with low-dose
chemotherapy (6-mercaptopurine and low-dose METHODS
cytarabine arabinoside) for disease control [4,5]. Patients and Diagnostic Criteria
Although the efficacy of azacitidine, a DNA meth- Patients were recruited for the JPLSG JMML-11
yltransferase inhibitor, has been reported trial (UMIN000005936) between July 2011 and
recently, most patients with JMML require alloge- June 2017. Eligibility criteria included a diagnosis
neic hematopoietic cell transplantation (HCT) as a of JMML according to international criteria [10]
curative therapy [6,7]. Busulfan (BU), an alkylating and age <14 years at diagnosis. Genetic diagnoses
agent, is widely used as a conditioning regimen were made based on the Sanger sequencing of
for HCT in patients with JMML. The European four RAS pathway genes (PTPN11, NRAS, KRAS, and
Working Group of Myelodysplastic Syndromes in CBL) and a clinical diagnosis of neurofibromatosis
children (EWOG-MDS) and the European Society type 1. Exclusion criteria included Noonan syn-
for Blood and Marrow Transplantation (EBMT) rec- drome, only germline RAS pathway gene muta-
ommend BU + cyclophosphamide (CY) + melphalan tions, and acute myeloid leukemia-type
(MEL) as the standard conditioning regimen before chemotherapy. Written informed consent was
HCT [8], resulting in overall survival (OS) and event- obtained from each patient’s guardian(s)
 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10 105.e3

according to the Declaration of Helsinki, and Insti- inhibitor (CsA or TAC) was tapered starting on day
tutional Review Board approval was obtained for 50 post-HCT and discontinued by day 180.
all aspects of the study.
Study Endpoints and Definitions
Treatment The primary study endpoint was EFS, defined as
All patients who met the eligibility criteria survival without graft failure, relapse, death from
(suitable donor identified and without organ dys- any causes, or second malignant neoplasm. Other
function or uncontrolled infections) underwent endpoints included OS, neutrophil engraftment,
HCT. The donor selection was made by the physi- acute and chronic GVHD, relapse, and transplanta-
cian in charge. The BU+FLU+MEL conditioning reg- tion-related mortality (TRM). Neutrophil engraft-
imen consisted of i.v. BU, 16 doses administered ment was defined as the first of 3 consecutive
every 6 hours, with dose adjustment based on PK days with an absolute neutrophil count
.5 £ 109/
studies on days -11 to -8; FLU, 30 mg/m2/day or 1 L. Chimerism of peripheral blood (every 2 weeks
mg/kg/day for patients <10 kg or age <1 year on up to day 100 and every 3 months after day 100
days -7 to -4; and MEL, 90 mg/m2/day or 3 mg/kg/ for 2 years) and bone marrow (BM) was evaluated
day for patients <10 kg or age <1 year on days -3 using short tandem repeat analyses. Acute and
to -2. chronic GVHD were diagnosed and graded
according to standard definitions [12,13]. TRM
was defined as all deaths without relapse or dis-
BU PK study ease progression. The incidence of acute GVHD
The BU PK study was performed as described was evaluated in patients with engraftment and
previously [11]. In brief, PK parameters were cal- chronic GVHD in engrafted patients who survived
culated using a one-compartment PK model. A for more than 100 days after HCT. HLA matching
test dose of BU (.6 mg/kg/dose) was administered was assessed using allelic DNA typing at the HLA-
to all 28 patients 1 week before initiating the con- A, -B, -C, and -DRB1 loci. Toxicity was evaluated
ditioning regimen. The initial BU dose was calcu- according to the Common Terminology Criteria
lated using the PK study results and a steady-state for Adverse Events v3.0.
concentration within 600 to 900 ng/mL was tar-
geted. In 23 of the 28 patients, the PK study was Statistical Analysis
repeated after the first BU dose during condition- EFS and OS probabilities were calculated using
ing, and the subsequent BU dose was adjusted Kaplan-Meier analysis, and groups were com-
according to the results. The total area under the pared using the log-rank test. Engraftment,
curve (AUC) was calculated retrospectively. All PK relapse, and TRM probabilities were estimated
samples were analyzed at the Department of using the cumulative incidence function, and
Pharmacy, Shinshu University Hospital, Nagano groups were compared using the Gray test. All
Prefecture, Japan. probabilities are expressed with 95% confidence
interval (CI). Competing events were death with-
GVHD Prophylaxis out engraftment for engraftment, death or relapse
GVHD in patients with an HLA-matched sibling without acute or chronic GVHD, death without
donor was treated prophylactically with cyclo- relapse for relapse, and relapse for TRM. Receiver
sporine A (CsA), 1.5 mg/kg twice daily, starting on operating characteristic curves were used to
day -1). GVHD in patients with an HLA-mis- determine the WBC count cutoff value for predict-
matched related donor (MMRD) or unrelated ing therapy outcomes. A P value <.05 was consid-
donor was treated with tacrolimus (TAC), .02 mg/ ered statistically significant. All statistical analyses
kg/day in a 24-hour continuous infusion, starting were performed using EZR 1.55 (Saitama Medical
on day -1, plus methotrexate (MTX), 15 mg/m2 on Center, Jichi Medical University, Saitama, Japan)
day 1 and 10 mg/m2/day on days 3, 6, and 11. For [14].
cord blood transplantation, one of the following
treatments was used to treat GVHD: TAC, .02 mg/ RESULTS
kg/day in a 24-hour continuous infusion starting Patients
on day -1, plus MTX, 10 mg/m2 on day 1, 7 mg/ Thirty-one patients with newly diagnosed
m2/day on days 3 and 6, or CsA, 1.5 mg/kg twice JMML were enrolled in this study. Enrollment was
daily, starting on day -1, plus MTX, 10 mg/m2 on declined by the attending physicians due to a
day 1, 7 mg/m2/day on days 3 and 6. In the change in the condition of 3 patients, who were
absence of grade
II acute GVHD, calcineurin excluded from further analyses (Figure 1). Patient
105.e4 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10

Figure 1. Flowchart describing eligible patients. Complete (100%) donor type chimerism was achieved in 19 of the 21 patients
who achieved neutrophil engraftment. Eighteen patients maintained complete remission, but 3 patients died of causes other
than leukemia (SOS, n = 1; bronchiolitis obliterans organizing pneumonia, n = 1; sudden death of unknown cause, n = 1). Three
patients experienced leukemia relapse, and 2 of these patients achieved long-term survival after a second HCT. HPS, hemopha-
gocytic syndrome, PD, progressive disease.

characteristics and transplantation procedures for Table 1

the remaining 28 patients are shown in Table 1. Patient and Transplantation Characteristics
Eighteen patients were males and 10 were
Characteristic Value
females. The median age at diagnosis and trans-
No. of eligible patients 28
plantation were 14 months (range, 0 to 48
Sex, n
months) and 23 months (range, 7 to 50 months),
respectively. The median interval between diag- Male 18
nosis and HCT was 6 months (range, 2 to 34 Female 10
months). Genetic analysis identified mutations in Age at diagnosis, mo, 14 (0-48)
median (range)
PTPN11 (n = 8), KRAS (n = 7), and NRAS (n = 4).
Two patients had clinical evidence of neurofibro- Age at HCT, mo, median (range) 23 (7-50)
matosis type 1. Seven patients showed no evi- BSA at HCT, m2, median (range) .48 (.36-.63)
dence of RAS pathway gene mutations. Interval between diagnosis and 6 (2
34)
HCT, mo, median (range)
PB at diagnosis, median (range)
Transplantation Procedure
WBC count, £ 109/L 20.7 (2.1-244.0)
A family donor was available for 7 patients (3
Monocyte count, £ 109/L 4.2 (.3-122.0)
HLA-matched siblings, 3 HLA 1-antigen- mis-
Platelet count, £ 109/L 45.5 (10.0-210.0)
matched parents, and 1 haploidentical father).
Overall, 21 patients received grafts from unrelated Blasts, % 2 (0-13)
donors, including 8 HLA-matched donors and 13 HbF, % 9 (1-68)
HLA-mismatched donors. The graft sources were PB at the beginning of conditioning,
median (range)
related BM for 7 patients, unrelated BM for 14
patients, and unrelated cord blood for 7 patients. WBC count, £ 109/L 4.1 (.3-14.5)
Monocyte count, £ 109/L .6 (0-3.7)
Platelet count, £ 109/L 33.5 (7.0-301.0)
Engraftment
Neutrophil engraftment (ANC
.5 £ 109/L) was Blasts, % 0 (0-18)
achieved in 21 of 28 patients (75%) at a median of Spleen size at diagnosis, cm, 4.5 (0-13.5)
median (range)
20.5 days (range, 11 to 39 days) after transplanta-
Spleen size at the beginning 4.0 (0-13.5)
tion. One patient died on day +27 from post-trans-
of conditioning,
plantation hemophagocytic syndrome and heart cm, median (range)
failure before engraftment. Six patients did not
(continued)
achieve neutrophil engraftment; 5 of these
 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10 105.e5

Table 1 (Continued) CI, 42% to 78%), 52% (95% CI, 32% to 69%), 18% (95%
Characteristic Value CI, 6% to 34%), and 21% (95% CI, 9% to 38%), respec-
Karyotype, n tively (Figure 2). The median follow-up of the 18
Normal 19
survivors was 4.4 years (range, 1.4 to 7.0 years).
The causes of death for the 10 patients who died
-7/7q- 4
are shown in Figure 1. The 100-day cumulative
Other abnormalities 5
incidences of acute grade II-IV and grade III-IV
RAS pathway mutation, n
acute GVHD were 62% (95% CI, 34% to 78%) and
PTPN11 8
38% (95% CI, 13% to 56%), respectively. The 1-year
KRAS 7
cumulative incidence of chronic GVHD was 16%
NRAS 4
(95% CI, 0 to 31%) (Supplementary Figure S1A, B).
NF1* 2
None 7 Toxicities Associated with Conditioning
Chemotherapy before HSCT, n Regimens
6MP 13 Nonhematologic adverse events occurring
6MP § CA § ETP 11 within 30 days post-HCT are summarized in Sup-
None 4 plementary Table S1. Febrile neutropenia, diar-
Donor, n rhea, hypoxemia, and mucositis occurred in 24
MSD 3 (86%), 11 (39%), 6 (21%), and 5 (18%) patients,
MMRD 4 respectively. BU-associated toxicities, including
MUD 8 SOS and thrombotic microangiopathy (TMA),
MMUD 13 occurred in 7 patients (25%; SOS, n = 6; TMA, n = 2).
Stem cell source, n
BM 21
CB 7 Univariable Analyses of Risk Factors for EFS, OS,
GVHD prophylaxis, n Relapse, TRM, and Engraftment
TAC-based 25 Results of univariable analyses to identify fac-
CsA-based 3
tors associated with EFS, OS, relapse, and TRM are
shown in Table 2. RAS pathway mutations, WBC
Conditioning drug dose, median (range)
count before the conditioning regimen
FLU actual dose, mg/m2 117 (78-124)
(
7.0 £ 109/L), and actual doses of FLU (<120 mg/
MEL actual dose, mg/m2 174 (117-181)
m2) and MEL (<180 mg/m2) were significantly
BU calculated total AUC, 72,960
associated with inferior EFS and OS. Body surface
ng・h/mLy (57,404-93,548)
area (BSA)
.5 m2, spleen size <4 cm before condi-
CA, cytarabine; CB, cord blood; ETP, etoposide; HbF, fetal
hemoglobin; MMRD, HLA-mismatched related donor;
tioning, and use of HLA-matched unrelated BM
MMUD, HLA-mismatched unrelated donor; MSD, HLA- donors were significantly associated with better
matched sibling donor; MUD, HLA-matched unrelated OS (Supplementary Figures S2 and S3). RAS path-
donor; PB, peripheral blood; 6MP, 6-mercaptopurine. way mutations and WBC count before conditioning
* Clinical evidence of NF1. (
7.0 £ 109/L) were risk factors for relapse, and
y
5 missing.
BSA <.5 m2 and abnormal karyotype were risk fac-
tors for TRM (Figures 3 and 4). Higher WBC count
before conditioning (
7.0 £ 109/L) was associated
patients underwent salvage second HCT, and 1 with a lower engraftment rate (Figure 3E).
patient achieved long-term survival. Of the 21
patients with neutrophil engraftment, 1 patient BU PK Study
died without platelet engraftment owing to sinu- We compared retrospectively calculated total
soidal obstruction syndrome (SOS). The other 20 AUC values for 23 patients with PK study data
patients achieved platelet engraftment of after the first BU dose, in patients with (n = 18)

20 £ 109/L, at a median of 41.5 days (range, 11 and without engraftment (n = 5) and in patients
to 116 days) after HCT. with (n = 6) and without SOS (n = 17) (Table 3).
No significant differences were found between
EFS, OS, Relapse, TRM, and GVHD, the 2 groups in either comparison. The remaining
The 1-year post-transplantation EFS was 57% 5 patients who did not undergo a PK study after
(95% CI, 37% to 73%). The 3-year OS, 3-year EFS, 3- the first BU dose did not develop SOS, and 2 of
year relapse rate, and 3-year TRM were 63% (95% these patients did not achieve engraftment.
105.e6 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10

Figure 2. Transplantation outcomes. (A) OS and EFS. (B) Relapse and TRM.

Table 2
Univariable Analysis of Influenced Factors for OS, EFS, Relapse, and TRM
Variable n 3-yr OS 3-yr EFS 3-yr Relapse 3-yr TRM
% 95% CI P % 95% CI P % 95% CI P % 95% CI P
Age at HCT
<2 yr 15 51 24-74 .212 44 19-68 .555 13 2-36 .537 33 11-57 .112

2 yr 13 77 44-92 62 31-82 23 5-49 8 0-30
BSA
<.5 m2 16 42 17-65 .014 35 13-59 .068 19 4-42 .917 38 15-61 .02

.5 m2 12 92 54-99 75 41-91 17 2-43 0 0-0
Karyotype
Normal 19 67 40-84 .356 51 26-71 .886 26 9-48 .102 11 2-29 .037
Abnormal 9 56 20-81 56 20-81 0 0-0 44 12-76
RAS pathway mutation
PTPN11 8 88 39-98 .005 63 23-86 .004 13 1-45 .005 13 1-45 .392
NF1 2 0 0-0 0 0-0 50 0-96 50 0-96
NRAS 4 25 1-67 25 1-67 25 0-77 50 2-88
KRAS 7 64 15-90 64 15-90 0 0-0 14 1-49
None 7 71 26-92 57 17-84 29 3-64 14 1-49
Time from diagnosis to HCT
<6 mo 16 68 38-85 .667 48 22-70 .793 13 2-34 .386 25 7-48 .515

6 mo 12 58 27-80 58 27-80 25 5-52 17 2-43
Chemotherapy before HCT
Yes 24 66 43-81 .444 53 31-71 .754 21 7-39 .346 17 5-34 .151
No 4 50 6-85 50 6-85 0 0-0 50 2-88
Donor source
CB 7 57 17-84 .802 29 4-61 .189 14 0-50 .005 29 3-64 .304
Related BM 7 57 17-84 43 10-73 57 13-86 0 0-0
Unrelated BM 14 71 41-88 71 41-88 0 0-0 29 8-53
HLA
Match 11 82 45-95 .146 73 37-90 .126 18 3-46 .915 9 0-35 .184
Mismatch 17 52 26-72 39 16-62 18 4-39 29 10-52
WBC count at start of conditioning
7.0 £ 109/L 21 75 49-89 .027 60 35-78 .045 10 2-27 .046 19 6-38 .658
>7.0 £ 109/L 7 29 4-61 29 4-61 43 7-77 29 3-64
Spleen size at start of conditioning
4.0 cm 16 81 53-94 .035 69 41-86 .052 13 2-34 .407 13 2-34 .219
>4.0 cm 12 38 11-65 28 6-57 25 5-52 33 9-60
FLU actual dose
<120 mg/m2 21 51 28-70 .038 37 17-57 .010 24 8-44 .113 29 11-49 .115

120 mg/m2 7 100 100-100 100 100-100 0 0-0 0 0-0
MEL actual dose
<180 mg/m2 19 47 24-67 .013 36 16-57 .014 21 6-42 .508 32 12-53 .064

180 mg/m2 9 100 100-100 89 43-98 11 1-41 0 0-0
BU calculated total AUC*
<73,000 ng・h/mL 11 51 18-77 .298 51 18-77 .508 9 0-35 .925 27 6-55 .851

73,000 ng・h/mL 12 75 41-91 67 34-86 8 0-33 25 5-52

* 5 missing.
 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10 105.e7

Figure 3. Effects of WBC counts at the beginning of conditioning on transplantation outcomes. (A, B) Patients with WBC
7.0 £ 109/L before conditioning (n = 21) exhibited significantly higher 3-year OS and EFS than patients with WBC
>7.0 £ 109/L (n = 7): 75% (95% CI, 49% to 89%) versus 27% (95% CI, 4% to 61%) and 60% (95% CI, 35% to 78%) versus 29% (95% CI,
4% to 61%), respectively. (C) The cumulative incidence of relapse at 3 years was significantly lower in patients with a WBC
7.0 £ 109/L than in patients with a WBC >7.0 £ 109/L: 10% (95% CI, 2% to 27%) versus 43% (95% CI, 7% to 77%). (D) No differen-
ces in 3-year TRM were observed: 19% (95% CI, 6% to 38%) versus 29% (95% CI, 3% to 64%). (E) The cumulative incidence of
engraftment at 60 days was significantly higher in patients with a WBC 7.0 £ 109/L compared to patients with a WBC
>7.0 £ 109/L.

Figure 4. Effect of BSA on transplantation outcomes. (A, B) The 3-year OS of patients with a BSA <.5 (n = 16) was significantly
better than that of patients with a BSA
.5 (n = 12): 42% (95% CI 17% to 65%) versus 92% (95% CI, 54% to 99%). (B, C) No differ-
ence in the 3-year EFS and relapse was observed: 35% (95% CI, 13% to 59%) versus 75% (95% CI, 41% to 91%) and 19% (95% CI, 4%
to 42%) versus 17% (95% CI, 2% to 43%). (D) The cumulative incidence of TRM at 3 years was significantly higher in patients
with a BSA <.5 compared to patients with a BSA
.5: 38% (95% CI, 15% to 61%) versus 0%. (E) No difference in engraftment at
60 days was observed: 69% (95% CI, 37% to 87%) versus 83% (95% CI, 41% to 96%).
105.e8 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10

Table 3
Busulfan PK Study
Parameter Engraftment SOS
(+) (-) P (+) (-) P
No. of patients 18 5 6 17
First-dose PK, average § SD
Vd, L/kg .761 § .179 .675 § .097 .248 .735 § .146 .745 § .177 .905
CL, L/kg/hr .256 § .077 .226 § .056 .436 .251 § .100 .248 § .064 .92
Css, ng/mL 769.8 § 165.8 708.7 § 134.7 .462 721.0 § 167.6 730.7 § 206.9 .924
Calculated total AUC, 75,332.5 § 9979.8 72,203.7 § 7829.3 .526 75,484.8 § 74,358.5 § .809
ng・h/mL, average § SD 6574.0 10,472.9
Vd indicates volume of distribution; CL, clearance; Css, steady state of concentration.

DISCUSSION be redefined for patients with smaller body sizes.

In the prospective multicenter JMML-11 study, In addition, FLU and MEL dosage adjustment
we evaluated the efficacy and safety of a BU+FLU based on PK studies has been reported to be useful
+MEL conditioning regimen in 28 JMML patients. [15,16] and is expected to be implemented in
The probabilities of engraftment, 3-year OS, EFS, future studies.
relapse, and TRM were 75%, 63%, 52%, 18%, and Tumor control with low-dose chemotherapy
21%, respectively. These results were consistent pretransplantation was recommended for
with a retrospective study using Japanese trans- patients included in the current study; however,
plant registry data that also showed favorable the study did not include patients treated with
outcomes for this regimen (86% 60-day engraft- AML-type chemotherapy and azacytidine, reflect-
ment, 73% 5-year OS, 59% 5-year EFS, 26% 5-year ing the standard of care for JMML at the time
relapse, and 9% 5-year TRM) [9]. The standard [5,17,18]. Clinical indicators associated with higher
conditioning regimen for patients with JMML pro- tumor burdens prior to HCT, including splenomeg-
posed by the EWOG-MDS and EBMT consists of aly (
4 cm) and higher WBC counts (
7.0 £ 109/
BU+CY+MEL, and the reported probabilities of sus- L), were significantly associated with worse OS.
tained engraftment, 4-year OS, EFS, relapse, and Notably, higher WBC count also was associated
TRM for this regimen are 95%, 64%, 52%, 35%, and with an inferior engraftment rate. These results are
13%, respectively [8]. Despite the relatively lower consistent with previous reports of better trans-
engraftment and higher TRM, these results indi- plantation outcomes in patients who underwent
cate that the BU+FLU+MEL regimen may provide pretransplantation AML chemotherapy or
similar survival outcomes as BU+CY+MEL and FLU + cytarabine to reduce the pretransplantation
may become one of the standard conditioning tumor burden [19
21]. A recent prospective clini-
regimens for patients with JMML. cal study from the EWOG demonstrated that azaci-
In the present study, patients with a BSA
.5 tidine provides valuable clinical benefits to JMML
m2 (n = 12) exhibited excellent transplantation patients before HCT [7]. Appropriate pretransplan-
outcomes, with a 3-year OS rate of 92% and a 3- tation tumor control with these bridging therapies
year TRM of 0%. Conversely, patients with a BSA may contribute to improved transplantation out-
<.5 m2 (n = 16) exhibited poor prognoses, with a comes for patients with JMML.
3-year OS rate of 42% and a 3-year TRM of 38%. In the current study, significantly better out-
Because the FLU and MEL doses were calculated comes with no relapses were achieved in patients
per kilogram for patients weighing <10 kg, it is receiving HLA-matched unrelated BM (n = 8) com-
possible that patients with a lower BSA received pared with other stem cell sources (Supplemen-
lower actual doses of these agents, resulting in tary Figure S2 and S3); thus, HLA-matched
worse prognoses. Although BU was appropriately unrelated BM may be the most appropriate choice
adjusted based on PK results, the cumulative inci- for transplantation using the BU+FLU+MEL regi-
dence of BU-related complications, including SOS men. Patients with JMML often experience a rapid
and TMA, was significantly higher in this group and aggressive clinical course, even after a consid-
(Supplementary Figure S1C). Therefore, BU+FLU erable period of stable disease [4,22]. Thus, it is
+MEL may be an appropriate conditioning regi- essential to initiate the search for an unrelated
men option for patients with JMML with BSA
.5 donor immediately after diagnosis in patients
m2, although the target AUC for BU may need to with JMML.
 K. Sakashita et al. / Transplantation and Cellular Therapy 30 (2024) 105.e1
105.e10 105.e9

The limitations of this study include the small myeloid neoplasms and acute leukemia. Blood. 2016;
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2. Niemeyer CM, Arico M, Basso G, et al. Chronic myelo-
justified because JMML is a rare leukemia. Future
monocytic leukemia in childhood: a retrospective anal-
analysis with large real-world data from interna- ysis of 110 cases. European Working Group on
tional transplant registries may provide a compar- Myelodysplastic Syndromes in Childhood (EWOG-
ative evaluation of the BU+FLU+MEL regimen MDS). Blood. 1997;89:3534–3543.
3. Niemeyer CM. RAS diseases in children. Haematologica.
versus the standard BU+CY+MEL regimen. In addi-
2014;99:1653–1662.
tion, next-generation sequencing or methylation 4. Locatelli F, Niemeyer CM. How I treat juvenile myelo-
analysis results were not available, because study monocytic leukemia. Blood. 2015;125:1083–1090.
enrollment began in 2011. Future clinical trials for 5. Bergstraesser E, Hasle H, Rogge T, et al. Non-hematopoi-
etic stem cell transplantation treatment of juvenile
JMML should include these molecular profiling
myelomonocytic leukemia: a retrospective analysis and
data. definition of response criteria. Pediatr Blood Cancer.
2007;49:629–633.
CONCLUSION 6. Cseh A, Niemeyer CM, Yoshimi A, et al. Bridging to
transplant with azacitidine in juvenile myelomonocytic
The results of this prospective multicenter leukemia: a retrospective analysis of the EWOG-MDS
study (JMML-11) confirm the positive results of study group. Blood. 2015;125:2311–2313.
previous retrospective analyses and suggest that 7. Niemeyer CM, Flotho C, Lipka DB, et al. Response to
BU+FLU+MEL might become one of the standard upfront azacitidine in juvenile myelomonocytic leuke-
mia in the AZA-JMML-001 trial. Blood Adv. 2021;5:
conditioning regimens for patients with JMML, 2901–2908.
except for children of small body size. 8. € llke P, Zecca M, et al. Hematopoietic stem
Locatelli F, No
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myelomonocytic leukemia (JMML): results of the
ACKNOWLEDGMENTS EWOG-MDS/EBMT trial. Blood. 2005;105:410–419.
The authors thank Enago (https://www.enago. 9. Yoshida N, Sakaguchi H, Yabe M, et al. Clinical outcomes
jp) for the English language review. after allogeneic hematopoietic stem cell transplantation
Financial disclosure: This study was supported in children with juvenile myelomonocytic leukemia: a
report from the Japan Society for Hematopoietic Cell
by a Grant for Clinical Cancer Research from the Transplantation. Biol Blood Marrow Transplant. 2020;
Ministry of Health, Labor, and Welfare of Japan 26:902–910.
(H20-GanRinsho-Ippan-017) and Grants for Practi- 10. Chan RJ, Cooper T, Kratz CP, Weiss B, Loh ML. Juvenile
cal Research for Innovative Cancer Control from myelomonocytic leukemia: a report from the 2nd Inter-
national JMML Symposium. Leuk Res. 2009;33:355–362.
the Japan Agency for Medical Research and Devel- 11. Takachi T, Arakawa Y, Nakamura H, et al. Personalized
opment (16ck0106064h0003, 17ck0106329h0001, pharmacokinetic targeting with busulfan in allogeneic
18ck0106329h0002, 19ck0106329h0003, and hematopoietic stem cell transplantation in infants with
21ck0106611h0002). acute lymphoblastic leukemia. Int J Hematol. 2019;110:
355–363.
Conflict of interest statement: There are no con- 12. Sullivan KM, Agura E, Anasetti C, et al. Chronic graft-
flicts of interest to report. versus-host disease and other late complications of
Authorship statement: K.S. and N.Y. designed bone marrow transplantation. Semin Hematol. 1991;28:
and performed the research, analyzed the data, 250–259.
13. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consen-
and wrote the manuscript. H.M. performed the sus Conference on Acute GVHD Grading. Bone Marrow
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script. Y.O., K.W., M.Y., H.K., Y.H., and A.M. 14. Kanda Y. Investigation of the freely available easy-to-
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project. K.M. performed the chimerism analysis. kinetics guided melphalan dose adjustment in reduced
M.H. and S.O. performed the BU PK study. T.W. intensity conditioning allogeneic transplant for non-
analyzed the data. All authors critically reviewed malignant disorders. Br J Clin Pharmacol. 2022;88:115–
127.
and revised the manuscript. 16. Fabrizio VA, Boelens JJ, Mauguen A, et al. Optimal flu-
darabine lymphodepletion is associated with improved
SUPPLEMENTARY MATERIALS outcomes after CAR T-cell therapy. Blood Adv. 2022;6:
1961–1968.
Supplementary material associated with this
17. Festa RS, Shende A, Lanzkowsky P. Juvenile chronic
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doi:10.1016/j.jtct.2023.10.002. tion chemotherapy. Med Pediatr Oncol. 1990;18:311–316.
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