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Blood First Edition Paper, prepublished online January 6, 2015; DOI 10.1182/blood-2014-08-550483
How I treat Juvenile Myelomonocytic Leukemia (JMML) 1,2
Franco Locatelli
3,4
and Charlotte M. Niemeyer
1
Department of Pediatric Hematology-Oncology, Istituto di Ricovero e Cura a Carattere Scientifico
(IRCCS) Bambino Gesù Children’s Hospital, Rome, Italy; 2
Department of Pediatrics, University of Pavia.
3
Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of Freiburg
Germany; 4
German Cancer Consortium (DKTK), Heidelberg, Germany and German Cancer Research Center
(DKFZ), Heidelberg, Germany
Address correspondence to: Prof. Franco Locatelli, University of Pavia, Department of Pediatric Hematology and Oncology IRCCS Ospedale Pediatrico Bambino Gesù, Piazza Sant'Onofrio, 4. 00165 Rome-Italy Phone: +39 06 68592678/2129 Fax:
+39 06 68592292
e-mail: [email protected]
Research grants: This work was partially supported by grants from AIRC (Associazione Italiana Ricerca sul Cancro, progetto speciale 5xmille), PRIN (Progetti di Rilevante Interesse Nazionale) 2010, MIUR (Ministero dell’Istruzione, Università e della Ricerca), Ministero della Salute (Ricerca Finalizzata 2010) e IRCCS Ospedale Pediatrico Bambino Gesù to F.L.
1 Copyright © 2015 American Society of Hematology
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Abstract Juvenile myelomonocytic leukemia (JMML) is a unique, aggressive hematopoietic disorder of infancy/early childhood, due to excessive proliferation of cells of monocytic and granulocytic lineages. Approximately 90% of patients carry either somatic or germline mutations of PTPN-11, K-
RAS, N-RAS, CBL or NF1 in their leukemic cells. These genetic aberrations are largely mutually exclusive and activate the Ras/mitogen-activated protein kinase (MAPK) pathway. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the therapy of choice for most of JMML patients, being able to cure more than 50% of affected children. We recommend that this option be promptly offered to any child with PTPN-11, K-RAS, NF1 mutated JMML and to the majority of those with N-RAS mutations. Since children with CBL mutations and few of those with N-RAS mutations may experience spontaneous resolution of hematological abnormalities, the decision to proceed to transplantation in these patients must be carefully weighed. Disease recurrence remains the main cause of treatment failure after HSCT. A second allograft is recommended if overt JMML relapse occurs after transplantation. Recently, azacytidine, a hypomethylating agent, was reported to induce hematological/molecular remissions in some JMML children and its role in both reducing leukemia burden before HSCT and in non-transplant settings requires further studies.
2
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Clinical case A
26-month-old
boy
was
referred
to
the
Pediatric
Department
because
of
fever,
lymphadenopathy, facial skin rash, abdominal distension, bruising and pallor. Spleen and liver were palpable 5 and 3 cm below the costal margin, respectively. Cell blood count showed 9
9
leukocytosis (58x10 /L), thrombocytopenia (29x10 /L), anemia (7 gr/dL) and striking monocytosis 9
(3,95x10 /L); dysplastic monocytes were observed at peripheral blood smear evaluation. The bone marrow (BM) aspirate revealed hypercellular marrow, with 7% blast cells. No bcr/abl fusion transcript was found and karyotype on BM cells was 46,XY. A diagnosis of juvenile myelomonocytic leukemia (JMML) was suspected and, then, confirmed by detection of somatic N-RAS mutation. Since no family HLA-identical donor was available, a search for locating a suitable either unrelated volunteer or cord blood (CB) unit was started. A 5/6 HLA-matched CB unit was located and, 3 months after diagnosis, the patient received umbilical CB transplantation (UCBT) after a busulfanbased myeloablative regimen. After initial detection of complete donor chimerism, mixed chimerism with 10% recipient cells was documented on day +51 during tapering of cyclosporine (Cs-A), which had been administered for graft-versus-host disease (GvHD) prophylaxis. CsA was immediately discontinued and, 10 days later, the child developed grade II acute GvHD, which resolved with steroid therapy. Complete donor chimerism was detected since day +90 after UCBT and, 3 years after transplantation, the child remains disease free.
This case illustrates some of the typical diagnostic/therapeutic features related to this rare myeloproliferative disease of early childhood. Indeed, JMML is characterized by overproduction of monocytic and granulocytic cells that infiltrate different organs, including spleen, liver, lung, and 1-4
gastro-intestinal tract, where diarrhea, sometimes with bloody features, may occur.
3
Affected
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children usually show pallor, fever, and skin bleeding, which are the results of anemia, leukocytosis and thrombocytopenia. In contrast to Philadelphia-positive chronic myeloid leukemia, 9
the white blood (WBC) count rarely exceeds 100x10 /L, the median value reported in a large 9
9
2
9
cohort of patients being 33x10 /L (range 5-259x10 /L). A WBC under 10x10 /L at diagnosis is 2
occasionally noted, particularly in children with monosomy 7.
Morphological evaluation of peripheral blood smear is the most important step in establishing the diagnosis. Immature monocytes, along with myelocytes, metamyelocytes and erythroblasts, are 1,2,5,6
usually found.
Almost all cases show striking monocytosis, with dysplastic cells; an absolute 9
4,5,7,8
monocyte count exceeding 1x10 /L is required for diagnosis of JMML.
A remarkable feature of
many JMML cases with normal karyotype is a markedly increased synthesis of fetal hemoglobin 9
(HbF). BM findings in JMML are not by themselves diagnostic, but rather consistent with the diagnosis. BM aspirate shows hypercellularity with predominance of granulocytic cells at all stages of maturation, although, occasionally, erythroid series predominates; blast percentage is moderately elevated, but never reaches the level seen in acute leukemia. Chromosomal studies of leukemic cells show monosomy 7 in approximately 25% of JMML patients, other abnormalities being found in another 10% of children; however, the majority (65%) 2,3,5,10
of cases has a normal karyotype.
The frequency of monosomy 7 among the genetic subtypes
varies, being, in our experience, more frequently detected in children with K-RAS anomalies. JMML may have an incidence of up to 1.2/million children per year, accounting for 2-3% of all 11
childhood hematological malignancies.
Median age at presentation is 2 years (range 0.1-11.4) 2
and males are more frequently affected (male/female ratio 2-3:1). To account for both the myelodysplastic and proliferative features noted in JMML, the World Health Organization (WHO) 12
classification placed the entity in the group of myelodysplastic/myeloproliferative disorders.
4
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13
Diagnosing children with JMML is sometimes challenging, since the clinical picture of JMML can 14
be mimicked by a number of human herpes-virus infections,
leukocyte-adhesion deficiency,
infantile malignant osteopetrosis, hemophagocytic lymphohistiocytosis and Wiskott-Aldrich 5,15
syndrome.
The recent discovery that approximately 90% of children with JMML have either
somatic or germline mutations in genes involved in the RAS signaling pathway (see below), besides greatly improving our understanding of the molecular pathogenesis of this disorder, has enormously facilitated diagnosis by allowing mutational analyses. It is not, therefore, surprising 8
that the criteria initially proposed and utilized for many years for diagnosing JMML have been revised over time.
4,5,7
The most recent version of the criteria where oncogenetic features largely
predominate and that we currently use to diagnose children with JMML is reported in Table 1.
Pathophysiology and genetics of JMML Hypersensitivity of JMML myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and pathological activation of the RAS-RAF-MAPK (mitogen-activated protein 16,17
kinase) signaling pathway play an important role in disease pathophysiology.
Indeed, GM-CSF
hypersensitivity of monocyte/macrophage colonies is a hallmark of the disease, and, for many 18,19
years, represented an important diagnostic tool.
This peculiar GM-CSF hypersensitivity can still
be utilized as diagnostic criterion for JMML for those few patients without a detectable known molecular lesion (see also Table 1). However, GM-CSF hypersensitivity has been reported also to 20
21
be induced by HHV-6 and cytomegalovirus infections, and the assay is not standardized among laboratories. Recently, hyper-phosphorylation of STAT5 in response to GM-CSF documented low
through phospho-specific flow cytometry in either CD33+/CD34+ or CD33+CD14+CD38 22,23
been found in a proportion of patients with JMML
and, thus, could also be helpful in
supporting the diagnosis of children without any detectable molecular lesion (Table 1).
5
cells has
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As already mentioned, major progress in understanding the pathogenesis of JMML has been 4,10,16,24
achieved in the last 2 decades by mapping out the underlying genetic lesions.
This
molecular characterization was certainly facilitated or even promoted by the discovery of a group of genetic syndromes resulting from germline mutations in genes of the RAS/MAPK pathways. These mutations induce the activation of the pathway, and thus, these disorders, collectively grouped as “neuro-cardio-facio cutaneous syndromes (NCFCS)” or “RASopathies”, share common clinical features, including propensity to develop malignancies, among which myeloproliferative 25,26
disorders are of relevant importance.
Neurofibromatosis type 1 (NF-1) and Noonan syndrome
(NS), due to mutations of NF1 the former and of PTPN-11 in half of the cases the latter, are the 25-27
most frequent and known ones.
A transient myeloproliferative disorder (TMD) is diagnosed in
a proportion of NS children in the neonatal period/early infancy. In contrast to JMML, NS/MPD generally resolves spontaneously over months; thus a “watch and wait” strategy is appropriate for 4,25
these children.
These infants with NS very often have cardiac anomalies in addition, and a
certain proportion of them may develop life-threatening or even fatal complications at least in part due to leukocytosis and tissue invasion by monocytes and immature granulocytes. We recommend that this subset of NS patients be treated with mild cytoreductive therapy, such as 6mercaptopurine. Moreover, since some cases of NS/MPD acquire cytogenetic abnormalities and progress to classically aggressive JMML, we recommend also that these patients be followed closely to timely diagnose and treat possible evolution. The spectrum of mutations described thus far in JMML occur in genes all encoding proteins that 10
signal through the RAS-RAF-MAPK pathways. and, as most recently demonstrated, CBL.
These genes include NF1, N-RAS, K-RAS, PTPN-11,
10,16,28
While largely mutually exclusive, mutations in
NF1, PTPN-11, K-RAS, N-RAS and CBL are detected in 90% of patients with JMML.16,24 Specifically, approximately, 35% of JMML patients carry somatic mutations in PTPN-11,
6
29,30
20-25% carry
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aberrations in N-RAS or K-RAS, 2,4,34
31-33
while 11% of patients with JMML were diagnosed with clinical
Since heterozygous point mutations noted in PTPN-11 and RAS may arise at a somatic
NF-1.
or germline level, we recommend that genetic screening in leukemic cells be followed by studies in non-hematopoietic tissue, such as fibroblasts, cells attached to nails, or hair bulbs. The reason for this recommendation lies on the observation that, besides those with NS and germline PTPN-11 mutations, the rare children carrying germline N-RAS and K-RAS mutations and presenting myeloproliferation generally show amelioration with time and, thus, do not require HSCT (Table 35
2).
Patients carrying CBL mutation (found in up to 15% of all cases) deserve a particular mention, since they display several congenital anomalies that overlap with those observed in NF-1, NS and Legius 28,36,37
syndrome.
These children have germline CBL mutations and develop JMML because they
are at increased risk of developing loss of heterozygosity (LOH) for the CBL locus in hematopoietic stem/progenitor cells. Indeed, all children with JMML and CBL mutations were found to have germline CBL missense mutation on one allele and acquired LOH on the other allele in leukemic cells. The resulting myeloproliferative disorder is often self-resolving, although these children may 28,36,38,39
have an aggressive clinical course (Figure 1).
Some children with germline CBL mutations
and JMML experienced vasculitis later in life. Similarly, mice that lacked Cbl developed severe vascular lesions with massive infiltration of T cells and high concentrations of anti-double stranded 40
DNA antibodies.
Although mutations in NF1, PTPN-11, K-RAS, N-RAS and CBL represent the most prominent genetic feature of JMML known so far, it is still unclear how they determine treatment resistance. Alterations of genes, such as JAK2, TET2, RUNX1, ASXL1, seen in other myeloproliferative disorders of adulthood are rare.
37,41,42
Recently, through an exome-sequencing approach, it was confirmed
that JMML is characterized by a paucity of gene mutations. Secondary mutations of SETBP1 and 7
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JAK3
43
were the most frequent ones, being detected in around 15% of children.
SETBP1
and
JAK3
These mutations of
were presumed to be involved in tumor progression and were associated with
43
poor clinical outcomes.
Recently, a study based on the use of
droplet digital polymerase chain
reaction (ddPCR) identified SETBP1 mutations in 17 out of 56 (30%) children with JMML 44 .
study,
SETBP1
Also in this
mutations were associated with a dismal prognosis.
Should all children with JMML be immediately offered allogeneic HSCT?
In the vast majority of cases, JMML is an aggressive and fatal disorder if left untreated, the median
2
survival time of children who do not receive an allograft being as short as 10-12 months.
Blastic
transformation is infrequent in JMML, and most untreated patients succumb to respiratory failure
due to pulmonary infiltration of leukemic cells. Clinical risk assessment in JMML includes age at
diagnosis,
platelet
2,6
variables.
platelet
2,6
and
percentage
of
HbF
adjusted
for
patient
age
as
main
prognostic
In particular, in a large cohort of JMML patients, age above 2 years at diagnosis,
count
survival.
count
below
British
9
33x10 /L,
investigators
and
levels
devised
9
a
of
HbF>10%
scoring
system,
were
the
where
main
HbF>10%
predictors
and
of
short
platelet
count
6
24 months), increased HbF (>10%), and, importantly, appeared to be an unfavorable
66
prognostic factor predicting relapse following HSCT.
How to optimize the transplant approach in children with JMML
12
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Preparative regimens that do not include total body irradiation (TBI) are particularly attractive for
children with JMML, since radiation-induced late effects may be especially deleterious for very
67-70
young
Moreover,
children.
myeloablative
children
with
therapy
JMML
offered
are
in
a
greater
prepared
retrospective
anti-leukemic
to
HSCT
using
EWOG-MDS
efficacy than
a
analysis,
47
TBI.
myeloablative
busulfan-based
Thus, we
regimen
advise
based
on
that
the
combination of busulfan with other cytotoxic drugs (Figure 2). The preparative regimen used by
48,57
the EWOG-MDS group includes busulfan, cyclophosphamide and melphalan.
The rationale for
this choice lies on the consideration that a preparative regimen consisting of three alkylating drugs
that
have
non-cell-cycle
specific
action
appears
potentially
capable
of
eradicating
stem
cell
disorders, such as JMML, in which a portion of clonogenic cells are dormant out of cell cycle.
Japanese
investigators
recently
reported
on
the
use
of
a
preparative
regimen
consisting
of
busulfan, fludarabine and melphalan in an attempt to decrease TRM and reduce the risk of graft
failure.
49
Although, the number of patients reported in this study is much smaller than that of the
EWOG-MDS
group,
at
the
time
of
reporting,
7/10
patients
transplanted
were
alive
and
in
remission, with a median follow-up of 30 months. In the recent retrospective analysis on 110
children
with
JMML
given
regimen
recommended
by
single-unit
the
57
therapy had similar outcome.
unrelated
EWOG-MDS
UBCT,
group
or
patients
another
given
either
the
busulfan-based
preparative
myeloablative
The Children’s Oncology Group (COG) is currently prospectively
comparing the preparative regimen including busulfan, cyclophosphamide and melphalan versus a
combination of busulfan and fludarabine, in order to test the hypothesis that the latter regimen is
associated with both less TRM and comparable DFS.
Serotherapy (such as anti-thymocyte globulin, ATG) is often included in the conditioning regimen
when an unrelated donor, either of BM, peripheral blood or CB, is employed. Despite theoretical
concerns that serotherapy may potentially reduce
13
in vivo
anti-leukemia alloreactivity, the available
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48,57
evidence indicates that its use does not increase the relapse rate of children with JMML.
By
contrast, ATG may contribute to reduce the risk of fatal complications related to GvHD. Thus, we
believe that ATG should be employed in those children with JMML given an unrelated donor HSCT
or UCBT.
Incidence of both acute and chronic GvHD in patients with JMML reported in the different studies
48,50,57
tend to be lower than in other leukemic diseases,
this being possibly related to the young
age of patients with JMML, and GvHD did not represent a major cause of TRM. In the study on
children with JMML given UCBT, decreased incidence of relapse was associated with the presence
57
of grade II-III acute GvHD.
better
survival
50,51
not.
These
for
children
findings
Moreover, investigators from the
who
suggest
did
the
develop
chronic
existence
of
a
GvHD
as
United States and Japan noted
compared
graft-versus-leukemia
with
those
who
did
(GvL)
effect
directed
against JMML cells with subsequent protection against relapse. Support to this hypothesis is given
by
the
observation
that
withdrawal
of
immunosuppressive
therapy
in
patients
71-73
relapse of JMML can prevent the subsequent occurrence of overt relapse.
considerations,
we
recommend
that
JMML
children
with
NF-1,
somatic
with
incipient
In view of all these
PTPN-11
or
N-RAS
mutations, older than 4 years at time of diagnosis or with more than 20% of blasts at time of HSCT,
receive low-intensity GvHD prophylaxis with the aim of optimizing the GvL effect (Figure 2). In the
absence of acute GvHD, prophylaxis should be discontinued between day +60 and +90 after HSCT.
A
remarkable
exception
to
this
recommendation
is
represented
by
children
carrying
K-RAS
mutations, as, in our experience, these children have lower relapse rate than children with other
molecular abnormalities (Figure 2).
Gross
spleen
enlargement
is
usually
found
in
many
children
47,74
splenectomy has been utilized in several children with JMML,
with
JMML.
Pre-transplantation
also with the hope of promoting
donor cell engraftment and of reducing tumor burden at time of HSCT, thus potentially translating
14
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into a reduced risk of recurrence. However, splenectomy before HSCT, as well as spleen size at
time of the
allograft, was not found
48
children with JMML.
to have any impact on post-transplantation outcome of
Thus, available data are not in favor of indiscriminate use of splenectomy
before transplantation, the potential advantages having to be weighed against the risks related to
75
the procedure or to post-splenectomy infections.
The presence of massive splenomegaly with
evidence of hypersplenism and/or refractoriness to platelet transfusions could be arguments for
considering this procedure in selected cases.
There is no doubt that a relevant area of controversy surrounding the care of JMML patients
concerns also the role of anti-leukemic therapy prior to transplantation. Indeed, the need or even
opportunity of treating JMML children with conventional chemotherapy is currently uncertain and
the comparative evaluation of different clinical protocols has been hampered by lack of uniform
76
criteria for response.
To date, no standard chemotherapy regimens used prior to HSCT have
been shown to have real impact on the incidence of post-HSCT relapse. In some patients, decrease
in
leukocytosis
and
cytarabine.
In
fludarabine
and
spleen
children
size
with
high-dose
can
blastic
be
noted
with
transformation
cytarabine
may
give
oral
or
6-mercaptopurine
life-threatening
temporary
relief.
A
and/or
pulmonary
watchful
low-dose
infiltration,
waiting
can
be
considered for those few patients with JMML who are asymptomatic. Overall, the pre-transplant
therapy is still a matter of controversy and firm recommendations cannot be provided (see also
Figure 1).
How we manage children experiencing disease recurrence after an allograft
The algorithm that we use for treating either incipient or overt leukemia relapse in patients either
still receiving or off any immune-suppressive therapy is reported in Figure 3. Serial quantitative
chimerism studies using short-tandem repeat (STR) markers have been shown to be useful for
15
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identifying
JMML
71,73
patients
with
increasing
mixed
chimerism
and,
therefore,
at
high
risk
of
In these patients, immediate withdrawal of on-going GvHD prophylaxis can lead to
relapse.
71-73
eradication of malignant cells re-growing/persisting after the conditioning regimen.
Although
77
effective, this strategy can, however, lead in some patients to development of chronic GvHD.
More sensitive methods to detect re-emergence/persistence of malignant cells, such as those
relying on disease-specific marker, may allow identification of a smaller percentage of residual
leukemic
cells,
72
withdrawal.
this
For
potentially
this
purpose,
increasing
a
the
chance
fluorescent-based,
assay for detecting the most common
RAS
or
PTPN-11
of
response
allele-specific
to
GvHD
polymerase
prophylaxis
chain
reaction
78
mutations has recently been developed.
Prospective studies aimed at validating the clinical benefit deriving from this approach are needed.
For
children
with
JMML
experiencing
overt
leukemia
relapse
71,79
lymphocyte infusion (DLI) proved to be largely ineffective,
after
allogeneic
HSCT,
donor
while a second allograft, from either
the same or a different donor, together with reduction of the intensity of GvHD prophylaxis aimed
at
optimizing
the
GvL
effect,
biological/immunological
72,79
obscure.
is
able
to
rescue
reasons
why
JMML
more
than
recurrence
has
one
third
limited
of
71,77,80
patients.
sensitivity
to
DLI
The
remain
Since a second HSCT can be an effective salvage therapy for children with JMML
71,77,80
relapsing after a first grafting procedure,
we do believe that children with JMML should not
receive DLI, but are to be given a second transplant as soon as possible, especially if they have
already discontinued GvHD prophylaxis.
What we envisage to be the role of innovative targeted drugs in JMML
So far, as already mentioned, therapies other than transplantation have had a limited role in
children with JMML;
76
also HSCT has some important limitations, including lack of efficacy in those
patients who relapse, TRM and long-term sequelae. Available data indicate that the Ras/MAPK
16
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pathway
is
deregulated
in
JMML
through
not
only
genetic
but
also
45,46,81
epigenetic
changes.
Whether the use of epigenetic drugs, such as 5-azacytidine (a DNA methyltransferase-inhibiting
azanucleoside
assumed
to
reverse
epigenetic
dysregulation
in
malignant
cells),
or
the
use
of
agents targeting the Ras/MAPK pathway prior to HSCT may be beneficial is currently unknown.
Furlan and colleagues reported a successful anecdotal case of azacytidine use in a JMML child with
both
K-RAS
82
mutation and monosomy 7.
The clinical and hematologic response obtained with the
st
drug was impressive; already after the 1
82
was noted.
th
After the 5
course, a response in spleen size and monocyte count
course, monosomy 7 disappeared, and the
th
diagnosis, became undetectable after 7 courses.
years
after
transplantation
the
child
remains
After the 8
disease
free.
K-RAS
mutation, present at
course, HSCT was performed, and 5
Since
this
case
was
reported,
we
observed clinical and molecular responses to 5-azacytidine in 3 out of 9 JMML patients treated off
label before transplantation (unpublished personal data). Despite its clinical activity, 5-azacytidine
is not expected to be ultimately curative in JMML; however, it can be tested as window therapy
with the aim of effectively reducing the burden of disease before HSCT. A phase-2, multicenter
study is underway in Europe to explore this hypothesis.
Analogous
studies
hematologic
evaluating
abnormalities
in
the
JMML
hypothesis
are
also
abrogated the myeloproliferative disease in
whether
worthy
Nf-1
and
of
Kras
MEK
inhibitors
consideration,
since
83-86
mutant mice.
might
MEK
ameliorate
inhibition
In particular, in
Kras
mutant mice, PD0325901, a highly selective pharmacological inhibitor of MEK, was shown to: i)
correct the aberrant proliferation and differentiation of bone marrow progenitor cells; ii) induce a
rapid and sustained reduction in leukocyte counts; iii) enhance erythropoiesis; and iv) prolong
24
mouse survival.
Future clinical studies aimed at testing whether treating children with JMML
with MEK inhibitors prior to HSCT might improve their clinical status by reducing the morbidity
caused by infiltration of organs with leukemic cells are warranted.
17
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Author contribution statement: Conflict of interest disclosure:
Both authors equally contributed to writing this manuscript.
Both authors do not have any conflict of interest to disclose.
18
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References 1. Arico M, Biondi A, Pui CH. Juvenile myelomonocytic leukemia. Blood. 1997;90(2):479-488. 2. Niemeyer CM, Arico M, Basso G, et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood. 1997;89(10):3534-3543. 3. Niemeyer CM, Kratz C. Juvenile myelomonocytic leukemia. Curr Oncol Rep. 2003;5(6):510-515. 4. Loh ML. Recent advances in the pathogenesis and treatment of juvenile myelomonocytic leukaemia. Br J Haematol. 2011;152(6):677-687. 5. Loh ML. Childhood myelodysplastic syndrome: focus on the approach to diagnosis and treatment of juvenile myelomonocytic leukemia. Hematology Am Soc Hematol Educ Program . 2010;2010:357-362. 6. Passmore SJ, Hann IM, Stiller CA, et al. Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood. 1995;85(7):1742-1750. 7. Chan RJ, Cooper T, Kratz CP, Weiss B, Loh ML. Juvenile myelomonocytic leukemia: a report from the 2nd International JMML Symposium. Leuk Res. 2009;33(3):355-362. 8. Niemeyer CM, Fenu S, Hasle H, Mann G, Stary J, van Wering E. Differentiating juvenile myelomonocytic leukemia from infectious disease - Response. Blood. 1998;91(1):365-367. 9. Weinberg RS, Leibowitz D, Weinblatt ME, Kochen J, Alter BP. Juvenile chronic myelogenous leukaemia: the only example of truly fetal (not fetal-like) erythropoiesis. Br J Haematol. 1990;76(2):307310. 10. Niemeyer CM, Kratz CP. Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia: molecular classification and treatment options. Br J Haematol. 2008;140(6):610-624. 11. Hasle H, Kerndrup G, Jacobsen BB. Childhood myelodysplastic syndrome in Denmark: incidence and predisposing conditions. Leukemia. 1995;9(9):1569-1572. 12. WHO Classification of tumors of haematopoietic and lymphoid tissues. . Lyon: IARC Press; 2008. 13. Pinkel D. Differentiating juvenile myelomonocytic leukemia from infectious disease. Blood. 1998;91(1):365-367. 14. Karow A, Baumann I, Niemeyer CM. Morphologic differential diagnosis of juvenile myelomonocytic leukemia--pitfalls apart from viral infection. J Pediatr Hematol Oncol. 2009;31(5):380. 15. Yoshimi A, Kamachi Y, Imai K, et al. Wiskott-Aldrich syndrome presenting with a clinical picture mimicking juvenile myelomonocytic leukaemia. Pediatr Blood Cancer. 2013;60(5):836-841. 16. de Vries AC, Zwaan CM, van den Heuvel-Eibrink MM. Molecular basis of juvenile myelomonocytic leukemia. Haematologica. 2010;95(2):179-182. 17. Kratz CP, Niemeyer CM. Juvenile myelomonocytic leukemia. Hematology. 2005;10 Suppl 1:100-103. 18. Emanuel PD, Bates LJ, Castleberry RP, Gualtieri RJ, Zuckerman KS. Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood . 1991;77(5):925-929. 19. Emanuel PD, Bates LJ, Zhu SW, Castleberry RP, Gualtieri RJ, Zuckerman KS. The role of monocytederived hemopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia. Exp Hematol. 1991;19(10):1017-1024. 20. Lorenzana A, Lyons H, Sawaf H, Higgins M, Carrigan D, Emanuel PD. Human herpesvirus 6 infection mimicking juvenile myelomonocytic leukemia in an infant. J Pediatr Hematol Oncol. 2002;24(2):136-141. 21. Moritake H, Ikeda T, Manabe A, Kamimura S, Nunoi H. Cytomegalovirus infection mimicking juvenile myelomonocytic leukemia showing hypersensitivity to granulocyte-macrophage colony stimulating factor. Pediatr Blood Cancer. 2009;53(7):1324-1326. 22. Hasegawa D, Bugarin C, Giordan M, et al. Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia. Blood Cancer J. 2013;3:e160. 19
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23. Kotecha N, Flores NJ, Irish JM, et al. Single-cell profiling identifies aberrant STAT5 activation in myeloid malignancies with specific clinical and biologic correlates. Cancer Cell. 2008;14(4):335-343. 24. Chang TY, Dvorak CC, Loh ML. Bedside to bench in juvenile myelomonocytic leukemia: insights into leukemogenesis from a rare pediatric leukemia. Blood. 2014. 25. Kratz CP, Rapisuwon S, Reed H, Hasle H, Rosenberg PS. Cancer in Noonan, Costello, cardiofaciocutaneous and LEOPARD syndromes. Am J Med Genet C Semin Med Genet . 2011;157C(2):83-89. 26. Kratz CP, Schubbert S, Bollag G, Niemeyer CM, Shannon KM, Zenker M. Germline mutations in components of the Ras signaling pathway in Noonan syndrome and related disorders. Cell Cycle. 2006;5(15):1607-1611. 27. Williams VC, Lucas J, Babcock MA, Gutmann DH, Korf B, Maria BL. Neurofibromatosis type 1 revisited. Pediatrics. 2009;123(1):124-133. 28. Niemeyer CM, Kang MW, Shin DH, et al. Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nat Genet. 2010;42(9):794-800. 29. Tartaglia M, Niemeyer CM, Fragale A, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet. 2003;34(2):148-150. 30. Kratz CP, Niemeyer CM, Castleberry RP, et al. The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease. Blood. 2005;106(6):21832185. 31. Miyauchi J, Asada M, Sasaki M, Tsunematsu Y, Kojima S, Mizutani S. Mutations of the N-ras gene in juvenile chronic myelogenous leukemia. Blood. 1994;83(8):2248-2254. 32. Flotho C, Valcamonica S, Mach-Pascual S, et al. RAS mutations and clonality analysis in children with juvenile myelomonocytic leukemia (JMML). Leukemia. 1999;13(1):32-37. 33. Kalra R, Paderanga DC, Olson K, Shannon KM. Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras. Blood. 1994;84(10):3435-3439. 34. Shannon KM, O'Connell P, Martin GA, et al. Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. N Engl J Med. 1994;330(9):597601. 35. De Filippi P, Zecca M, Lisini D, et al. Germ-line mutation of the NRAS gene may be responsible for the development of juvenile myelomonocytic leukaemia. Br J Haematol. 2009;147(5):706-709. 36. Loh ML, Sakai DS, Flotho C, et al. Mutations in CBL occur frequently in juvenile myelomonocytic leukemia. Blood. 2009;114(9):1859-1863. 37. Muramatsu H, Makishima H, Jankowska AM, et al. Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia. Blood. 2010;115(10):1969-1975. 38. Matsuda K, Taira C, Sakashita K, et al. Long-term survival after nonintensive chemotherapy in some juvenile myelomonocytic leukemia patients with CBL mutations, and the possible presence of healthy persons with the mutations. Blood. 2010;115(26):5429-5431. 39. Perez B, Mechinaud F, Galambrun C, et al. Germline mutations of the CBL gene define a new genetic syndrome with predisposition to juvenile myelomonocytic leukaemia. J Med Genet. 2010;47(10):686-691. 40. Naramura M, Jang IK, Kole H, Huang F, Haines D, Gu H. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat Immunol . 2002;3(12):1192-1199. 41. Zecca M, Bergamaschi G, Kratz C, et al. JAK2 V617F mutation is a rare event in juvenile myelomonocytic leukemia. Leukemia. 2007;21(2):367-369. 42. Perez B, Kosmider O, Cassinat B, et al. Genetic typing of CBL, ASXL1, RUNX1, TET2 and JAK2 in juvenile myelomonocytic leukaemia reveals a genetic profile distinct from chronic myelomonocytic leukaemia. Br J Haematol. 2010;151(5):460-468. 43. Sakaguchi H, Okuno Y, Muramatsu H, et al. Exome sequencing identifies secondary mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia. Nat Genet. 2013;45(8):937-941. 44. Stieglitz E, Troup CB, Gelston LC, et al. Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia. Blood. 2014. 20
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45. Olk-Batz C, Poetsch AR, Nollke P, et al. Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome. Blood. 2011;117(18):4871-4880. 46. Poetsch AR, Lipka DB, Witte T, et al. RASA4 is a target of DNA hypermethylation in resistant juvenile myelomonocytic leukemia. Epigenetics. 2014. 47. Locatelli F, Niemeyer C, Angelucci E, et al. Allogeneic bone marrow transplantation for chronic myelomonocytic leukemia in childhood: a report from the European Working Group on Myelodysplastic Syndrome in Childhood. J Clin Oncol. 1997;15(2):566-573. 48. Locatelli F, Nollke P, Zecca M, et al. Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOG-MDS/EBMT trial. Blood. 2005;105(1):410419. 49. Yabe M, Sako M, Yabe H, et al. A conditioning regimen of busulfan, fludarabine, and melphalan for allogeneic stem cell transplantation in children with juvenile myelomonocytic leukemia. Pediatr Transplant. 2008;12(8):862-867. 50. Manabe A, Okamura J, Yumura-Yagi K, et al. Allogeneic hematopoietic stem cell transplantation for 27 children with juvenile myelomonocytic leukemia diagnosed based on the criteria of the International JMML Working Group. Leukemia . 2002;16(4):645-649. 51. Smith FO, King R, Nelson G, et al. Unrelated donor bone marrow transplantation for children with juvenile myelomonocytic leukaemia. Br J Haematol . 2002;116(3):716-724. 52. Dvorak CC, Loh ML. Juvenile myelomonocytic leukemia: molecular pathogenesis informs current approaches to therapy and hematopoietic cell transplantation. Front Pediatr. 2014;2:25. 53. Matsuda K, Shimada A, Yoshida N, et al. Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations. Blood . 2007;109(12):54775480. 54. Matsuda K, Yoshida N, Miura S, et al. Long-term haematological improvement after non-intensive or no chemotherapy in juvenile myelomonocytic leukaemia and poor correlation with adult myelodysplasia spliceosome-related mutations. Br J Haematol. 2012;157(5):647-650. 55. Flotho C, Kratz CP, Bergstrasser E, et al. Genotype-phenotype correlation in cases of juvenile myelomonocytic leukemia with clonal RAS mutations. Blood. 2008;111(2):966-967; author reply 967-968. 56. Yusuf U, Frangoul HA, Gooley TA, et al. Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: the Seattle experience. Bone Marrow Transplant. 2004;33(8):805-814. 57. Locatelli F, Crotta A, Ruggeri A, et al. Analysis of risk factors influencing outcomes after cord blood transplantation in children with juvenile myelomonocytic leukemia: a EUROCORD, EBMT, EWOG-MDS, CIBMTR study. Blood. 2013;122(12):2135-2141. 58. Bertaina A, Bernardo ME, Caniglia M, Vinti L, Giorgiani G, Locatelli F. Cord blood transplantation in children with haematological malignancies. Best Pract Res Clin Haematol. 2010;23(2):189-196. 59. Locatelli F. Improving cord blood transplantation in children. Br J Haematol. 2009;147(2):217-226. 60. Rocha V, Locatelli F. Searching for alternative hematopoietic stem cell donors for pediatric patients. Bone Marrow Transplant . 2008;41(2):207-214. 61. Eapen M, Rubinstein P, Zhang MJ, et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study. Lancet. 2007;369(9577):1947-1954. 62. Barker JN, Davies SM, DeFor T, Ramsay NK, Weisdorf DJ, Wagner JE. Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair analysis. Blood. 2001;97(10):2957-2961. 63. Kurtzberg J, Prasad VK, Carter SL, et al. Results of the Cord Blood Transplantation Study (COBLT): clinical outcomes of unrelated donor umbilical cord blood transplantation in pediatric patients with hematologic malignancies. Blood. 2008;112(10):4318-4327. 64. Eapen M, Klein JP, Ruggeri A, et al. Impact of allele-level HLA matching on outcomes after myeloablative single unit umbilical cord blood transplantation for hematologic malignancy. Blood. 2014;123(1):133-140. 21
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65. Bresolin S, Zecca M, Flotho C, et al. Gene expression-based classification as an independent predictor of clinical outcome in juvenile myelomonocytic leukemia. J Clin Oncol. 2010;28(11):1919-1927. 66. Yoshida N, Yagasaki H, Xu Y, et al. Correlation of clinical features with the mutational status of GMCSF signaling pathway-related genes in juvenile myelomonocytic leukemia. Pediatr Res. 2009;65(3):334340. 67. Giorgiani G, Bozzola M, Locatelli F, et al. Role of busulfan and total body irradiation on growth of prepubertal children receiving bone marrow transplantation and results of treatment with recombinant human growth hormone. Blood. 1995;86(2):825-831. 68. Bernard F, Auquier P, Herrmann I, et al. Health status of childhood leukemia survivors who received hematopoietic cell transplantation after BU or TBI: an LEA study. Bone Marrow Transplant . 2014;49(5):709716. 69. Kunkele A, Engelhard M, Hauffa BP, et al. Long-term follow-up of pediatric patients receiving total body irradiation before hematopoietic stem cell transplantation and post-transplant survival of >2 years. Pediatr Blood Cancer. 2013;60(11):1792-1797. 70. Berbis J, Michel G, Chastagner P, et al. A French cohort of childhood leukemia survivors: impact of hematopoietic stem cell transplantation on health status and quality of life. Biol Blood Marrow Transplant . 2013;19(7):1065-1072. 71. Inagaki J, Fukano R, Nishikawa T, et al. Outcomes of immunological interventions for mixed chimerism following allogeneic stem cell transplantation in children with juvenile myelomonocytic leukemia. Pediatr Blood Cancer. 2013;60(1):116-120. 72. Locatelli F, Lucarelli B. Treatment of disease recurrence after allogeneic hematopoietic stem cell transplantation in children with juvenile myelomonocytic leukemia: a great challenge still to be won. Pediatr Blood Cancer. 2013;60(1):1-2. 73. Yoshimi A, Niemeyer CM, Bohmer V, et al. Chimaerism analyses and subsequent immunological intervention after stem cell transplantation in patients with juvenile myelomonocytic leukaemia. Br J Haematol. 2005;129(4):542-549. 74. Bunin N, Saunders F, Leahey A, Doyle J, Calderwood S, Freedman MH. Alternative donor bone marrow transplantation for children with juvenile myelomonocytic leukemia. J Pediatr Hematol Oncol. 1999;21(6):479-485. 75. Di Sabatino A, Carsetti R, Corazza GR. Post-splenectomy and hyposplenic states. Lancet. 2011;378(9785):86-97. 76. Bergstraesser E, Hasle H, Rogge T, et al. Non-hematopoietic stem cell transplantation treatment of juvenile myelomonocytic leukemia: a retrospective analysis and definition of response criteria. Pediatr Blood Cancer. 2007;49(5):629-633. 77. Chang YH, Jou ST, Lin DT, Lu MY, Lin KH. Second allogeneic hematopoietic stem cell transplantation for juvenile myelomonocytic leukemia: case report and literature review. J Pediatr Hematol Oncol. 2004;26(3):190-193. 78. Archambeault S, Flores NJ, Yoshimi A, et al. Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia. Blood. 2008;111(3):1124-1127. 79. Yoshimi A, Bader P, Matthes-Martin S, et al. Donor leukocyte infusion after hematopoietic stem cell transplantation in patients with juvenile myelomonocytic leukemia. Leukemia . 2005;19(6):971-977. 80. Yoshimi A, Mohamed M, Bierings M, et al. Second allogeneic hematopoietic stem cell transplantation (HSCT) results in outcome similar to that of first HSCT for patients with juvenile myelomonocytic leukemia. Leukemia. 2007;21(3):556-560. 81. Wlodarski MW, Motter J, Gorr TA, et al. Abnormal promoter DNA methylation in juvenile myelomonocytic leukemia is not caused by mutation in DNMT3A. Blood. 2011;118(16):4490-4491. 82. Furlan I, Batz C, Flotho C, et al. Intriguing response to azacitidine in a patient with juvenile myelomonocytic leukemia and monosomy 7. Blood. 2009;113(12):2867-2868. 83. Lauchle JO, Kim D, Le DT, et al. Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature . 2009;461(7262):411-414. 84. Chang T, Krisman K, Theobald EH, et al. Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice. J Clin Invest. 2013;123(1):335-339. 22
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85. Lyubynska N, Gorman MF, Lauchle JO, et al. A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice. Sci Transl Med . 2011;3(76):76ra27. 86. Gandre-Babbe S, Paluru P, Aribeana C, et al. Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemia. Blood . 2013;121(24):49254929.
23
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Table 1 Updated clinical and laboratory diagnostic criteria of JMML I.
Clinical and hematological features (all four features mandatory)
9
•
Peripheral blood monocyte count > 1x10 /L
•
Blast percentage in peripheral blood and bone marrow < 20%
•
Splenomegaly
•
Absence of Philadelphia chromosome (BCR/ABL rearrangement)
II. Oncogenetic studies (1 finding is sufficient)
PTPN11#
Somatic mutation in
•
Clinical diagnosis of NF-1 or germline
•
Germline
CBL
or
K-RAS#
•
or
NF1
N-RAS#*
mutation
mutation and loss of heterozygosity of
CBL**
III. Only for those patients (10% of the whole number) without any oncogenetic parameter, besides the clinical and haematological features listed under I, at least 2 of the following criteria have to be fulfilled:
-
Monosomy 7 or any other chromosomal abnormality Hemoglobin F increased for age Myeloid precursors on peripheral blood smear Spontaneous growth or GM-CSF hypersensitivity in colony assay Hyperphosphorilation of STAT5
# Germline mutations (indicating Noonan syndrome) need to be excluded.
Diagnosis of JMML but spontaneous regression of myeloproliferation may be noted in:
*
**
Few patients with
Patients with
CBL
N-RAS
mutation and normal HbF
germline mutation and loss of heterozygosity
24
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Table 2
Indication for HSCT in genetic subgroups of JMML
PTPN11
K-RAS
Noonan
Noonan
syndrome
syndrome
N-RAS
NF1
CBL
Noonan syndrome
CBL
Neurofibromatosis
syndrome
type 1
Germline mutations
(+/- LOH)
“watch and wait” “watch and wait” (mild
(mild
chemotherapy)
chemotherapy)
“watch and wait” (mild
“watch and HSCT
wait”
chemotherapy) HSCT if disease progression occurs
Somatic mutations
HSCT
HSCT
HSCT for most
LOH: loss of heterozygosity; HSCT: hematopoietic stem cell transplantation
25
-
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Figure Legends
Figure 1.
Algorithm for treatment of children with JMML, stratified according to somatic (N-RAS,
PTPN-11)
or germline (CBL,
some patients with somatic
NF1)
K-RAS,
molecular lesions and availability of an HLA-identical sibling. *In
N-RAS
mutations (i.e. those with low haemoglobin F and high-platelet
count), long-term survival in the absence of allogeneic hematopoietic stem cell transplantation (HSCT) has been observed. Although it is still an experimental therapy for JMML, haploidentical HSCT is an option in those few patients in need of an urgent allograft who lack any suitable HLAidentical sibling, unrelated adult donor or unrelated CB unit.
Figure 2.
Tailoring of graft-versus-host disease (GvHD) prophylaxis according to patient’s genetic lesions and other risks factors influencing the relapse rate. HSCT = hematopoietic stem cell transplantation; Bu = busulfan.
Figure 3.
Algorithm for treatment of mixed chimerism and leukemia relapse in JMML in patients either still receiving or off any immune-suppressive therapy. HSCT = hematopoietic stem cell transplantation.
26
Clinical signs of JMML
Figure 1
Molecular characterization Confirmed diagnosis (see also criteria in Table I)
NF1, PTPN11 KRAS, NRAS*, normal findings HLA-identical sibling
Start search UD/CBT
CBL
Watch and Wait Treatment at physician’s discretion
UD/CB unit available?
HSCT
Yes
HSCT
No
HaploHSCT
Consider HSCT if disease progression
Figure 2
HSCT Bu-Based Myeloablative regimen
NF-1, PTPN-11, N-RAS, no known molecular lesion and Age >4 years or Blasts % at HSCT >20%
K-RAS
High-intensity GVHD prophylaxis Low-intensity GVHD prophylaxis
Figure 3
HSCT - Mixed chimerism - Reappearence of cytogenetic/molecular alterations
On immunosuppressive (IS) therapy
Off immunosuppressive (IS) therapy
Immediate withdrawal of IS therapy
Complete Donor Chimerism Watch and Wait
Persistent Worsening
Second HSCT
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Prepublished online January 6, 2015; doi:10.1182/blood-2014-08-550483
How I treat juvenile myelomonocytic leukemia (JMML) Franco Locatelli and Charlotte M. Niemeyer
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Blood First Edition Paper, prepublished online January 6, 2015; DOI 10.1182/blood-2014-08-550483
How I treat Juvenile Myelomonocytic Leukemia (JMML) 1,2
Franco Locatelli
3,4
and Charlotte M. Niemeyer
1
Department of Pediatric Hematology-Oncology, Istituto di Ricovero e Cura a Carattere Scientifico
(IRCCS) Bambino Gesù Children’s Hospital, Rome, Italy; 2
Department of Pediatrics, University of Pavia.
3
Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of Freiburg
Germany; 4
German Cancer Consortium (DKTK), Heidelberg, Germany and German Cancer Research Center
(DKFZ), Heidelberg, Germany
Address correspondence to: Prof. Franco Locatelli, University of Pavia, Department of Pediatric Hematology and Oncology IRCCS Ospedale Pediatrico Bambino Gesù, Piazza Sant'Onofrio, 4. 00165 Rome-Italy Phone: +39 06 68592678/2129 Fax:
+39 06 68592292
e-mail: [email protected]
Research grants: This work was partially supported by grants from AIRC (Associazione Italiana Ricerca sul Cancro, progetto speciale 5xmille), PRIN (Progetti di Rilevante Interesse Nazionale) 2010, MIUR (Ministero dell’Istruzione, Università e della Ricerca), Ministero della Salute (Ricerca Finalizzata 2010) e IRCCS Ospedale Pediatrico Bambino Gesù to F.L.
1 Copyright © 2015 American Society of Hematology
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Abstract Juvenile myelomonocytic leukemia (JMML) is a unique, aggressive hematopoietic disorder of infancy/early childhood, due to excessive proliferation of cells of monocytic and granulocytic lineages. Approximately 90% of patients carry either somatic or germline mutations of PTPN-11, K-
RAS, N-RAS, CBL or NF1 in their leukemic cells. These genetic aberrations are largely mutually exclusive and activate the Ras/mitogen-activated protein kinase (MAPK) pathway. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the therapy of choice for most of JMML patients, being able to cure more than 50% of affected children. We recommend that this option be promptly offered to any child with PTPN-11, K-RAS, NF1 mutated JMML and to the majority of those with N-RAS mutations. Since children with CBL mutations and few of those with N-RAS mutations may experience spontaneous resolution of hematological abnormalities, the decision to proceed to transplantation in these patients must be carefully weighed. Disease recurrence remains the main cause of treatment failure after HSCT. A second allograft is recommended if overt JMML relapse occurs after transplantation. Recently, azacytidine, a hypomethylating agent, was reported to induce hematological/molecular remissions in some JMML children and its role in both reducing leukemia burden before HSCT and in non-transplant settings requires further studies.
2
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Clinical case A
26-month-old
boy
was
referred
to
the
Pediatric
Department
because
of
fever,
lymphadenopathy, facial skin rash, abdominal distension, bruising and pallor. Spleen and liver were palpable 5 and 3 cm below the costal margin, respectively. Cell blood count showed 9
9
leukocytosis (58x10 /L), thrombocytopenia (29x10 /L), anemia (7 gr/dL) and striking monocytosis 9
(3,95x10 /L); dysplastic monocytes were observed at peripheral blood smear evaluation. The bone marrow (BM) aspirate revealed hypercellular marrow, with 7% blast cells. No bcr/abl fusion transcript was found and karyotype on BM cells was 46,XY. A diagnosis of juvenile myelomonocytic leukemia (JMML) was suspected and, then, confirmed by detection of somatic N-RAS mutation. Since no family HLA-identical donor was available, a search for locating a suitable either unrelated volunteer or cord blood (CB) unit was started. A 5/6 HLA-matched CB unit was located and, 3 months after diagnosis, the patient received umbilical CB transplantation (UCBT) after a busulfanbased myeloablative regimen. After initial detection of complete donor chimerism, mixed chimerism with 10% recipient cells was documented on day +51 during tapering of cyclosporine (Cs-A), which had been administered for graft-versus-host disease (GvHD) prophylaxis. CsA was immediately discontinued and, 10 days later, the child developed grade II acute GvHD, which resolved with steroid therapy. Complete donor chimerism was detected since day +90 after UCBT and, 3 years after transplantation, the child remains disease free.
This case illustrates some of the typical diagnostic/therapeutic features related to this rare myeloproliferative disease of early childhood. Indeed, JMML is characterized by overproduction of monocytic and granulocytic cells that infiltrate different organs, including spleen, liver, lung, and 1-4
gastro-intestinal tract, where diarrhea, sometimes with bloody features, may occur.
3
Affected
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children usually show pallor, fever, and skin bleeding, which are the results of anemia, leukocytosis and thrombocytopenia. In contrast to Philadelphia-positive chronic myeloid leukemia, 9
the white blood (WBC) count rarely exceeds 100x10 /L, the median value reported in a large 9
9
2
9
cohort of patients being 33x10 /L (range 5-259x10 /L). A WBC under 10x10 /L at diagnosis is 2
occasionally noted, particularly in children with monosomy 7.
Morphological evaluation of peripheral blood smear is the most important step in establishing the diagnosis. Immature monocytes, along with myelocytes, metamyelocytes and erythroblasts, are 1,2,5,6
usually found.
Almost all cases show striking monocytosis, with dysplastic cells; an absolute 9
4,5,7,8
monocyte count exceeding 1x10 /L is required for diagnosis of JMML.
A remarkable feature of
many JMML cases with normal karyotype is a markedly increased synthesis of fetal hemoglobin 9
(HbF). BM findings in JMML are not by themselves diagnostic, but rather consistent with the diagnosis. BM aspirate shows hypercellularity with predominance of granulocytic cells at all stages of maturation, although, occasionally, erythroid series predominates; blast percentage is moderately elevated, but never reaches the level seen in acute leukemia. Chromosomal studies of leukemic cells show monosomy 7 in approximately 25% of JMML patients, other abnormalities being found in another 10% of children; however, the majority (65%) 2,3,5,10
of cases has a normal karyotype.
The frequency of monosomy 7 among the genetic subtypes
varies, being, in our experience, more frequently detected in children with K-RAS anomalies. JMML may have an incidence of up to 1.2/million children per year, accounting for 2-3% of all 11
childhood hematological malignancies.
Median age at presentation is 2 years (range 0.1-11.4) 2
and males are more frequently affected (male/female ratio 2-3:1). To account for both the myelodysplastic and proliferative features noted in JMML, the World Health Organization (WHO) 12
classification placed the entity in the group of myelodysplastic/myeloproliferative disorders.
4
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13
Diagnosing children with JMML is sometimes challenging, since the clinical picture of JMML can 14
be mimicked by a number of human herpes-virus infections,
leukocyte-adhesion deficiency,
infantile malignant osteopetrosis, hemophagocytic lymphohistiocytosis and Wiskott-Aldrich 5,15
syndrome.
The recent discovery that approximately 90% of children with JMML have either
somatic or germline mutations in genes involved in the RAS signaling pathway (see below), besides greatly improving our understanding of the molecular pathogenesis of this disorder, has enormously facilitated diagnosis by allowing mutational analyses. It is not, therefore, surprising 8
that the criteria initially proposed and utilized for many years for diagnosing JMML have been revised over time.
4,5,7
The most recent version of the criteria where oncogenetic features largely
predominate and that we currently use to diagnose children with JMML is reported in Table 1.
Pathophysiology and genetics of JMML Hypersensitivity of JMML myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and pathological activation of the RAS-RAF-MAPK (mitogen-activated protein 16,17
kinase) signaling pathway play an important role in disease pathophysiology.
Indeed, GM-CSF
hypersensitivity of monocyte/macrophage colonies is a hallmark of the disease, and, for many 18,19
years, represented an important diagnostic tool.
This peculiar GM-CSF hypersensitivity can still
be utilized as diagnostic criterion for JMML for those few patients without a detectable known molecular lesion (see also Table 1). However, GM-CSF hypersensitivity has been reported also to 20
21
be induced by HHV-6 and cytomegalovirus infections, and the assay is not standardized among laboratories. Recently, hyper-phosphorylation of STAT5 in response to GM-CSF documented low
through phospho-specific flow cytometry in either CD33+/CD34+ or CD33+CD14+CD38 22,23
been found in a proportion of patients with JMML
and, thus, could also be helpful in
supporting the diagnosis of children without any detectable molecular lesion (Table 1).
5
cells has
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As already mentioned, major progress in understanding the pathogenesis of JMML has been 4,10,16,24
achieved in the last 2 decades by mapping out the underlying genetic lesions.
This
molecular characterization was certainly facilitated or even promoted by the discovery of a group of genetic syndromes resulting from germline mutations in genes of the RAS/MAPK pathways. These mutations induce the activation of the pathway, and thus, these disorders, collectively grouped as “neuro-cardio-facio cutaneous syndromes (NCFCS)” or “RASopathies”, share common clinical features, including propensity to develop malignancies, among which myeloproliferative 25,26
disorders are of relevant importance.
Neurofibromatosis type 1 (NF-1) and Noonan syndrome
(NS), due to mutations of NF1 the former and of PTPN-11 in half of the cases the latter, are the 25-27
most frequent and known ones.
A transient myeloproliferative disorder (TMD) is diagnosed in
a proportion of NS children in the neonatal period/early infancy. In contrast to JMML, NS/MPD generally resolves spontaneously over months; thus a “watch and wait” strategy is appropriate for 4,25
these children.
These infants with NS very often have cardiac anomalies in addition, and a
certain proportion of them may develop life-threatening or even fatal complications at least in part due to leukocytosis and tissue invasion by monocytes and immature granulocytes. We recommend that this subset of NS patients be treated with mild cytoreductive therapy, such as 6mercaptopurine. Moreover, since some cases of NS/MPD acquire cytogenetic abnormalities and progress to classically aggressive JMML, we recommend also that these patients be followed closely to timely diagnose and treat possible evolution. The spectrum of mutations described thus far in JMML occur in genes all encoding proteins that 10
signal through the RAS-RAF-MAPK pathways. and, as most recently demonstrated, CBL.
These genes include NF1, N-RAS, K-RAS, PTPN-11,
10,16,28
While largely mutually exclusive, mutations in
NF1, PTPN-11, K-RAS, N-RAS and CBL are detected in 90% of patients with JMML.16,24 Specifically, approximately, 35% of JMML patients carry somatic mutations in PTPN-11,
6
29,30
20-25% carry
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aberrations in N-RAS or K-RAS, 2,4,34
31-33
while 11% of patients with JMML were diagnosed with clinical
Since heterozygous point mutations noted in PTPN-11 and RAS may arise at a somatic
NF-1.
or germline level, we recommend that genetic screening in leukemic cells be followed by studies in non-hematopoietic tissue, such as fibroblasts, cells attached to nails, or hair bulbs. The reason for this recommendation lies on the observation that, besides those with NS and germline PTPN-11 mutations, the rare children carrying germline N-RAS and K-RAS mutations and presenting myeloproliferation generally show amelioration with time and, thus, do not require HSCT (Table 35
2).
Patients carrying CBL mutation (found in up to 15% of all cases) deserve a particular mention, since they display several congenital anomalies that overlap with those observed in NF-1, NS and Legius 28,36,37
syndrome.
These children have germline CBL mutations and develop JMML because they
are at increased risk of developing loss of heterozygosity (LOH) for the CBL locus in hematopoietic stem/progenitor cells. Indeed, all children with JMML and CBL mutations were found to have germline CBL missense mutation on one allele and acquired LOH on the other allele in leukemic cells. The resulting myeloproliferative disorder is often self-resolving, although these children may 28,36,38,39
have an aggressive clinical course (Figure 1).
Some children with germline CBL mutations
and JMML experienced vasculitis later in life. Similarly, mice that lacked Cbl developed severe vascular lesions with massive infiltration of T cells and high concentrations of anti-double stranded 40
DNA antibodies.
Although mutations in NF1, PTPN-11, K-RAS, N-RAS and CBL represent the most prominent genetic feature of JMML known so far, it is still unclear how they determine treatment resistance. Alterations of genes, such as JAK2, TET2, RUNX1, ASXL1, seen in other myeloproliferative disorders of adulthood are rare.
37,41,42
Recently, through an exome-sequencing approach, it was confirmed
that JMML is characterized by a paucity of gene mutations. Secondary mutations of SETBP1 and 7
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JAK3
43
were the most frequent ones, being detected in around 15% of children.
SETBP1
and
JAK3
These mutations of
were presumed to be involved in tumor progression and were associated with
43
poor clinical outcomes.
Recently, a study based on the use of
droplet digital polymerase chain
reaction (ddPCR) identified SETBP1 mutations in 17 out of 56 (30%) children with JMML 44 .
study,
SETBP1
Also in this
mutations were associated with a dismal prognosis.
Should all children with JMML be immediately offered allogeneic HSCT?
In the vast majority of cases, JMML is an aggressive and fatal disorder if left untreated, the median
2
survival time of children who do not receive an allograft being as short as 10-12 months.
Blastic
transformation is infrequent in JMML, and most untreated patients succumb to respiratory failure
due to pulmonary infiltration of leukemic cells. Clinical risk assessment in JMML includes age at
diagnosis,
platelet
2,6
variables.
platelet
2,6
and
percentage
of
HbF
adjusted
for
patient
age
as
main
prognostic
In particular, in a large cohort of JMML patients, age above 2 years at diagnosis,
count
survival.
count
below
British
9
33x10 /L,
investigators
and
levels
devised
9
a
of
HbF>10%
scoring
system,
were
the
where
main
HbF>10%
predictors
and
of
short
platelet
count
6
24 months), increased HbF (>10%), and, importantly, appeared to be an unfavorable
66
prognostic factor predicting relapse following HSCT.
How to optimize the transplant approach in children with JMML
12
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Preparative regimens that do not include total body irradiation (TBI) are particularly attractive for
children with JMML, since radiation-induced late effects may be especially deleterious for very
67-70
young
Moreover,
children.
myeloablative
children
with
therapy
JMML
offered
are
in
a
greater
prepared
retrospective
anti-leukemic
to
HSCT
using
EWOG-MDS
efficacy than
a
analysis,
47
TBI.
myeloablative
busulfan-based
Thus, we
regimen
advise
based
on
that
the
combination of busulfan with other cytotoxic drugs (Figure 2). The preparative regimen used by
48,57
the EWOG-MDS group includes busulfan, cyclophosphamide and melphalan.
The rationale for
this choice lies on the consideration that a preparative regimen consisting of three alkylating drugs
that
have
non-cell-cycle
specific
action
appears
potentially
capable
of
eradicating
stem
cell
disorders, such as JMML, in which a portion of clonogenic cells are dormant out of cell cycle.
Japanese
investigators
recently
reported
on
the
use
of
a
preparative
regimen
consisting
of
busulfan, fludarabine and melphalan in an attempt to decrease TRM and reduce the risk of graft
failure.
49
Although, the number of patients reported in this study is much smaller than that of the
EWOG-MDS
group,
at
the
time
of
reporting,
7/10
patients
transplanted
were
alive
and
in
remission, with a median follow-up of 30 months. In the recent retrospective analysis on 110
children
with
JMML
given
regimen
recommended
by
single-unit
the
57
therapy had similar outcome.
unrelated
EWOG-MDS
UBCT,
group
or
patients
another
given
either
the
busulfan-based
preparative
myeloablative
The Children’s Oncology Group (COG) is currently prospectively
comparing the preparative regimen including busulfan, cyclophosphamide and melphalan versus a
combination of busulfan and fludarabine, in order to test the hypothesis that the latter regimen is
associated with both less TRM and comparable DFS.
Serotherapy (such as anti-thymocyte globulin, ATG) is often included in the conditioning regimen
when an unrelated donor, either of BM, peripheral blood or CB, is employed. Despite theoretical
concerns that serotherapy may potentially reduce
13
in vivo
anti-leukemia alloreactivity, the available
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48,57
evidence indicates that its use does not increase the relapse rate of children with JMML.
By
contrast, ATG may contribute to reduce the risk of fatal complications related to GvHD. Thus, we
believe that ATG should be employed in those children with JMML given an unrelated donor HSCT
or UCBT.
Incidence of both acute and chronic GvHD in patients with JMML reported in the different studies
48,50,57
tend to be lower than in other leukemic diseases,
this being possibly related to the young
age of patients with JMML, and GvHD did not represent a major cause of TRM. In the study on
children with JMML given UCBT, decreased incidence of relapse was associated with the presence
57
of grade II-III acute GvHD.
better
survival
50,51
not.
These
for
children
findings
Moreover, investigators from the
who
suggest
did
the
develop
chronic
existence
of
a
GvHD
as
United States and Japan noted
compared
graft-versus-leukemia
with
those
who
did
(GvL)
effect
directed
against JMML cells with subsequent protection against relapse. Support to this hypothesis is given
by
the
observation
that
withdrawal
of
immunosuppressive
therapy
in
patients
71-73
relapse of JMML can prevent the subsequent occurrence of overt relapse.
considerations,
we
recommend
that
JMML
children
with
NF-1,
somatic
with
incipient
In view of all these
PTPN-11
or
N-RAS
mutations, older than 4 years at time of diagnosis or with more than 20% of blasts at time of HSCT,
receive low-intensity GvHD prophylaxis with the aim of optimizing the GvL effect (Figure 2). In the
absence of acute GvHD, prophylaxis should be discontinued between day +60 and +90 after HSCT.
A
remarkable
exception
to
this
recommendation
is
represented
by
children
carrying
K-RAS
mutations, as, in our experience, these children have lower relapse rate than children with other
molecular abnormalities (Figure 2).
Gross
spleen
enlargement
is
usually
found
in
many
children
47,74
splenectomy has been utilized in several children with JMML,
with
JMML.
Pre-transplantation
also with the hope of promoting
donor cell engraftment and of reducing tumor burden at time of HSCT, thus potentially translating
14
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into a reduced risk of recurrence. However, splenectomy before HSCT, as well as spleen size at
time of the
allograft, was not found
48
children with JMML.
to have any impact on post-transplantation outcome of
Thus, available data are not in favor of indiscriminate use of splenectomy
before transplantation, the potential advantages having to be weighed against the risks related to
75
the procedure or to post-splenectomy infections.
The presence of massive splenomegaly with
evidence of hypersplenism and/or refractoriness to platelet transfusions could be arguments for
considering this procedure in selected cases.
There is no doubt that a relevant area of controversy surrounding the care of JMML patients
concerns also the role of anti-leukemic therapy prior to transplantation. Indeed, the need or even
opportunity of treating JMML children with conventional chemotherapy is currently uncertain and
the comparative evaluation of different clinical protocols has been hampered by lack of uniform
76
criteria for response.
To date, no standard chemotherapy regimens used prior to HSCT have
been shown to have real impact on the incidence of post-HSCT relapse. In some patients, decrease
in
leukocytosis
and
cytarabine.
In
fludarabine
and
spleen
children
size
with
high-dose
can
blastic
be
noted
with
transformation
cytarabine
may
give
oral
or
6-mercaptopurine
life-threatening
temporary
relief.
A
and/or
pulmonary
watchful
low-dose
infiltration,
waiting
can
be
considered for those few patients with JMML who are asymptomatic. Overall, the pre-transplant
therapy is still a matter of controversy and firm recommendations cannot be provided (see also
Figure 1).
How we manage children experiencing disease recurrence after an allograft
The algorithm that we use for treating either incipient or overt leukemia relapse in patients either
still receiving or off any immune-suppressive therapy is reported in Figure 3. Serial quantitative
chimerism studies using short-tandem repeat (STR) markers have been shown to be useful for
15
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identifying
JMML
71,73
patients
with
increasing
mixed
chimerism
and,
therefore,
at
high
risk
of
In these patients, immediate withdrawal of on-going GvHD prophylaxis can lead to
relapse.
71-73
eradication of malignant cells re-growing/persisting after the conditioning regimen.
Although
77
effective, this strategy can, however, lead in some patients to development of chronic GvHD.
More sensitive methods to detect re-emergence/persistence of malignant cells, such as those
relying on disease-specific marker, may allow identification of a smaller percentage of residual
leukemic
cells,
72
withdrawal.
this
For
potentially
this
purpose,
increasing
a
the
chance
fluorescent-based,
assay for detecting the most common
RAS
or
PTPN-11
of
response
allele-specific
to
GvHD
polymerase
prophylaxis
chain
reaction
78
mutations has recently been developed.
Prospective studies aimed at validating the clinical benefit deriving from this approach are needed.
For
children
with
JMML
experiencing
overt
leukemia
relapse
71,79
lymphocyte infusion (DLI) proved to be largely ineffective,
after
allogeneic
HSCT,
donor
while a second allograft, from either
the same or a different donor, together with reduction of the intensity of GvHD prophylaxis aimed
at
optimizing
the
GvL
effect,
biological/immunological
72,79
obscure.
is
able
to
rescue
reasons
why
JMML
more
than
recurrence
has
one
third
limited
of
71,77,80
patients.
sensitivity
to
DLI
The
remain
Since a second HSCT can be an effective salvage therapy for children with JMML
71,77,80
relapsing after a first grafting procedure,
we do believe that children with JMML should not
receive DLI, but are to be given a second transplant as soon as possible, especially if they have
already discontinued GvHD prophylaxis.
What we envisage to be the role of innovative targeted drugs in JMML
So far, as already mentioned, therapies other than transplantation have had a limited role in
children with JMML;
76
also HSCT has some important limitations, including lack of efficacy in those
patients who relapse, TRM and long-term sequelae. Available data indicate that the Ras/MAPK
16
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pathway
is
deregulated
in
JMML
through
not
only
genetic
but
also
45,46,81
epigenetic
changes.
Whether the use of epigenetic drugs, such as 5-azacytidine (a DNA methyltransferase-inhibiting
azanucleoside
assumed
to
reverse
epigenetic
dysregulation
in
malignant
cells),
or
the
use
of
agents targeting the Ras/MAPK pathway prior to HSCT may be beneficial is currently unknown.
Furlan and colleagues reported a successful anecdotal case of azacytidine use in a JMML child with
both
K-RAS
82
mutation and monosomy 7.
The clinical and hematologic response obtained with the
st
drug was impressive; already after the 1
82
was noted.
th
After the 5
course, a response in spleen size and monocyte count
course, monosomy 7 disappeared, and the
th
diagnosis, became undetectable after 7 courses.
years
after
transplantation
the
child
remains
After the 8
disease
free.
K-RAS
mutation, present at
course, HSCT was performed, and 5
Since
this
case
was
reported,
we
observed clinical and molecular responses to 5-azacytidine in 3 out of 9 JMML patients treated off
label before transplantation (unpublished personal data). Despite its clinical activity, 5-azacytidine
is not expected to be ultimately curative in JMML; however, it can be tested as window therapy
with the aim of effectively reducing the burden of disease before HSCT. A phase-2, multicenter
study is underway in Europe to explore this hypothesis.
Analogous
studies
hematologic
evaluating
abnormalities
in
the
JMML
hypothesis
are
also
abrogated the myeloproliferative disease in
whether
worthy
Nf-1
and
of
Kras
MEK
inhibitors
consideration,
since
83-86
mutant mice.
might
MEK
ameliorate
inhibition
In particular, in
Kras
mutant mice, PD0325901, a highly selective pharmacological inhibitor of MEK, was shown to: i)
correct the aberrant proliferation and differentiation of bone marrow progenitor cells; ii) induce a
rapid and sustained reduction in leukocyte counts; iii) enhance erythropoiesis; and iv) prolong
24
mouse survival.
Future clinical studies aimed at testing whether treating children with JMML
with MEK inhibitors prior to HSCT might improve their clinical status by reducing the morbidity
caused by infiltration of organs with leukemic cells are warranted.
17
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Author contribution statement: Conflict of interest disclosure:
Both authors equally contributed to writing this manuscript.
Both authors do not have any conflict of interest to disclose.
18
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Table 1 Updated clinical and laboratory diagnostic criteria of JMML I.
Clinical and hematological features (all four features mandatory)
9
•
Peripheral blood monocyte count > 1x10 /L
•
Blast percentage in peripheral blood and bone marrow < 20%
•
Splenomegaly
•
Absence of Philadelphia chromosome (BCR/ABL rearrangement)
II. Oncogenetic studies (1 finding is sufficient)
PTPN11#
Somatic mutation in
•
Clinical diagnosis of NF-1 or germline
•
Germline
CBL
or
K-RAS#
•
or
NF1
N-RAS#*
mutation
mutation and loss of heterozygosity of
CBL**
III. Only for those patients (10% of the whole number) without any oncogenetic parameter, besides the clinical and haematological features listed under I, at least 2 of the following criteria have to be fulfilled:
-
Monosomy 7 or any other chromosomal abnormality Hemoglobin F increased for age Myeloid precursors on peripheral blood smear Spontaneous growth or GM-CSF hypersensitivity in colony assay Hyperphosphorilation of STAT5
# Germline mutations (indicating Noonan syndrome) need to be excluded.
Diagnosis of JMML but spontaneous regression of myeloproliferation may be noted in:
*
**
Few patients with
Patients with
CBL
N-RAS
mutation and normal HbF
germline mutation and loss of heterozygosity
24
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Table 2
Indication for HSCT in genetic subgroups of JMML
PTPN11
K-RAS
Noonan
Noonan
syndrome
syndrome
N-RAS
NF1
CBL
Noonan syndrome
CBL
Neurofibromatosis
syndrome
type 1
Germline mutations
(+/- LOH)
“watch and wait” “watch and wait” (mild
(mild
chemotherapy)
chemotherapy)
“watch and wait” (mild
“watch and HSCT
wait”
chemotherapy) HSCT if disease progression occurs
Somatic mutations
HSCT
HSCT
HSCT for most
LOH: loss of heterozygosity; HSCT: hematopoietic stem cell transplantation
25
-
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Figure Legends
Figure 1.
Algorithm for treatment of children with JMML, stratified according to somatic (N-RAS,
PTPN-11)
or germline (CBL,
some patients with somatic
NF1)
K-RAS,
molecular lesions and availability of an HLA-identical sibling. *In
N-RAS
mutations (i.e. those with low haemoglobin F and high-platelet
count), long-term survival in the absence of allogeneic hematopoietic stem cell transplantation (HSCT) has been observed. Although it is still an experimental therapy for JMML, haploidentical HSCT is an option in those few patients in need of an urgent allograft who lack any suitable HLAidentical sibling, unrelated adult donor or unrelated CB unit.
Figure 2.
Tailoring of graft-versus-host disease (GvHD) prophylaxis according to patient’s genetic lesions and other risks factors influencing the relapse rate. HSCT = hematopoietic stem cell transplantation; Bu = busulfan.
Figure 3.
Algorithm for treatment of mixed chimerism and leukemia relapse in JMML in patients either still receiving or off any immune-suppressive therapy. HSCT = hematopoietic stem cell transplantation.
26
Clinical signs of JMML
Figure 1
Molecular characterization Confirmed diagnosis (see also criteria in Table I)
NF1, PTPN11 KRAS, NRAS*, normal findings HLA-identical sibling
Start search UD/CBT
CBL
Watch and Wait Treatment at physician’s discretion
UD/CB unit available?
HSCT
Yes
HSCT
No
HaploHSCT
Consider HSCT if disease progression
Figure 2
HSCT Bu-Based Myeloablative regimen
NF-1, PTPN-11, N-RAS, no known molecular lesion and Age >4 years or Blasts % at HSCT >20%
K-RAS
High-intensity GVHD prophylaxis Low-intensity GVHD prophylaxis
Figure 3
HSCT - Mixed chimerism - Reappearence of cytogenetic/molecular alterations
On immunosuppressive (IS) therapy
Off immunosuppressive (IS) therapy
Immediate withdrawal of IS therapy
Complete Donor Chimerism Watch and Wait
Persistent Worsening
Second HSCT
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Prepublished online January 6, 2015; doi:10.1182/blood-2014-08-550483
How I treat juvenile myelomonocytic leukemia (JMML) Franco Locatelli and Charlotte M. Niemeyer
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