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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3617-3623
RAPID COMMUNICATION
Myeloid Malignancies Induced by Alkylating Agents in Nf1 Mice
By
Nidal Mahgoub,
Brigit R. Taylor,
Michelle M. Le Beau,
Mary Gratiot,
Katrin M. Carlson,
Susan K. Atwater,
Tyler Jacks, and
Kevin M. Shannon
From the Departments of Pediatrics and Laboratory Medicine,
University of California, San Francisco, CA; the Section of
Hematology/Oncology, the Department of Medicine, University of Chicago,
Chicago, IL; and the Howard Hughes Medical Institute, the Department of
Biology, Massachusetts Institute of Technology, Cambridge, MA.
 |
ABSTRACT |
Therapy-related acute myeloid leukemia and myelodysplastic syndrome
(t-AML and MDS) are severe late complications of treatment with
genotoxic chemotherapeutic agents. Children with neurofibromatosis type
1 (NF1) are predisposed to malignant myeloid disorders that are
associated with inactivation of the NF1 tumor suppressor gene in the leukemic clone. Recent clinical data suggest that NF1 might be
also associated with an increased risk of t-AML after treatment with
alkyating agents. To test this hypothesis, we administered cyclophosphamide or etoposide to cohorts of wild-type and heterozygous Nf1 knockout mice. Cyclophosphamide exposure cooperated
strongly with heterozygous inactivation of Nf1 in myeloid
leukemogenesis, while etoposide did not. Somatic loss of the normal
Nf1 allele correlated with clinical disease and was more common
in 129/Sv mice than in 129/Sv × C57BL/6 animals. Leukemic cells
showing loss of heterozygosity at Nf1 retained a structural
allele on each chromosome 11 homolog. These studies establish a novel
in vivo model of alkylator-induced myeloid malignancy that will
facilitate mechanistic and translational studies.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IN 1977, ROWLEY ET AL1
described a group of patients who developed a myelodysplastic syndrome
(MDS) or acute myeloid leukemia (AML) as a second malignant neoplasm
after treatment for another cancer. Two forms of therapy-related (t)
myeloid malignancies are now recognized; these subtypes are associated
with distinct clinical and biologic features and differ with respect to
the prior anticancer treatment. Most patients with t-AML and t-MDS previously received chemotherapeutic agents that alkylate DNA. This
subtype typically involves a latency of 3 to 7 years between genotoxin
exposure and disease onset, a myelodysplastic prodrome, and frequent
loss of chromosomes 5 and/or 7 ( 5 and/or 7) or deletions
involving the long arms of these chromosomes
[del(5q)/del(7q)].1-4 The second subtype of t-AML
develops after therapy with drugs that inhibit topoisomerase II. These
cases are characterized by a shorter interval between cytotoxic therapy
and clinical signs, overt leukemia at presentation, and balanced
translocations that usually involve the MLL gene located on
chromosome 11, band q23.5-8 The prognosis is poor for
patients with t-AML or t-MDS. Importantly, as aggressive multi-agent
regimens are used increasingly to treat many primary cancers, the
incidence of therapy-related myeloid malignancies is expected to
increase over the next few years.
Individuals with neurofibromatosis type 1 (NF1) are predisposed to
specific benign and malignant neoplasms, which arise primarily in cells
derived from the embryonic neural crest.9 In addition, children (but not adults) with NF1 show a 200 to 500-fold increase in
the incidence of de novo malignant myeloid disorders, particularly juvenile myelomonocytic leukemia (JMML).10-12 The
NF1 gene encodes neurofibromin, a guanosine triphosphatase
(GTPase) activating protein that accelerates the slow
intrinsic rate of GTP hydrolysis on p21ras (Ras)
proteins.13 Genetic and biochemical data strongly support the hypothesis that NF1 functions as a tumor suppressor gene in human and murine hematopoietic cells by negatively regulating Ras
output.13-20 For example, approximately 10% of
heterozygous Nf1 knockout mice (Nf1+/) spontaneously
develop myeloid leukemia beginning around age 15 months, with tumor
cells exhibiting loss of the wild-type Nf1
allele.16
A few cases of t-AML associated with monosomy 7 were recently reported
in children with NF1, all of whom received alkylating agents to treat
primary cancers including anaplastic astrocytoma, glioblastoma, Wilms'
tumor, or acute lymphoblastic leukemia.21 However, because
these patients were not ascertained in a systematic way, it is
uncertain if the risk of therapy-related myeloid malignancies is
increased over children without NF1 who received similar therapies. Moreover, loss of heterozygosity (LOH) at NF1 was not detected in the leukemic cells of children with NF1 who developed
t-AML.21 If germline inactivation of NF1 cooperates
with genotoxic agents that are used to treat human cancers in
leukemogenesis, we reasoned that exposing Nf1+/ mice to
these drugs might produce an in vivo model of therapy-induced myeloid
disease. To test this hypothesis, we administered two chemotherapeutic
agents that have been implicated in human t-AML and t-MDS to
Nf1 mice. Here we show that the alkylating agent
cyclophosphamide, but not the toposiomerase II inhibitor etoposide,
efficiently induces a myeloproliferative disorder (MPD) in
Nf1+/ mice, and we present correlative cytogenetic and
molecular data.
| |
MATERIALS AND METHODS |
Animal care.
Mice were housed in the University of California, San Francisco (UCSF)
Animal Care facility and were examined regularly by one of the
investigators. Cyclophosphamide and etoposide were prepared by the UCSF
pharmacy and were administered by one of the investigators. Mice were
weighed at the beginning of the study and weekly thereafter to adjust
the drug doses. Complete blood counts (CBCs) were performed on blood
samples collected from tail veins in an automated cell counter. The
accuracy of abnormal blood counts was verified by direct examination of
stained smears. The study procedures were reviewed and approved by the
UCSF Committee for Animal Research.
Treatment and monitoring.
We mated Nf+/ and wild-type (Nf+/+) mice and
genotyped the offspring by Southern blot analysis of tail DNA. The
initial group of mice were from the inbred 129/Sv strain in which the
Nf1 mutation was created.16 To perform LOH analysis
at loci other than Nf1 in alkylator-treated mice, F1 offspring
of a cross between the 129/Sv and C57BL/6 strains were used in the
latter part of the study. Nf1+/ and Nf1+/+
littermates were assigned to observation (control group) or to receive
treatment with either etoposide or cyclophosphamide beginning at 6 to
10 weeks of age. These agents were selected because they are used
widely in human cancer therapy. Treated mice received a single 6-week
course of 100 mg/kg/wk of either agent, a schedule which approaches the
maximally tolerated doses. Cyclophosphamide (CY) was administered by
intraperitoneal injection whereas etoposide was administered through an
orogastric tube.
CBCs with white blood cell (WBC) differentials were performed every 3 months in mice that appeared well, and whenever a mouse showed signs of
systemic illness. The CBC was repeated immediately whenever the WBC
count was >20,000/µL. All mice that appeared moribund
and animals with WBC counts >20,000/µL on two consecutive determinations were killed, the spleens were weighed, and hematopoietic tissues were collected for morphologic and genetic analysis.
Nf1 genotyping and LOH analysis.
Genomic DNA was prepared from tail clippings or from hematopoietic
tissues (spleen or bone marrow) by standard procedures.22 Nf1 genotypes and loss of heterozygosity were determined by
digesting DNA samples with NcoI + HindIII followed by
gel electrophoresis, blotting to nylon membranes, and hybridization
with an NcoI-PstI fragment from intron 31 of
Nf1 as described previously.16 LOH was scored by
comparing the relative intensities of restriction fragments derived
from paired normal and leukemic tissues.
LOH analysis with microsatellite markers.
These procedures have been described in detail.14 Briefly,
DNA samples were amplified in a DNA Thermocycle Machine (Perkin Elmer
Cetus, Norwalk, CT). Polymerase chain reaction (PCR) was performed in reaction mixtures that include 0.66 µmol/L of respective 3' and 5' primers, 100 ng of target genomic DNA, 1 U of Taq polymerase (AmpliTaq; PE Applied Biosystems, Foster City, CA), and 0.4 µmol/L final concentrations of deoxynucleotides in a final reaction volume of
25 µL. The forward primer was kinase-labeled with
 -33P adenosine triphosphate (ATP). Labeled
PCR products were separated on (6 mol/L urea, 8% polyacylamide)
sequencing-type gels and run at 60 to 80 W constant power for 2 to 4 hours. The gels were dried, placed in Saran wrap (Dow Brands L.P.,
Indianapolis, IN), and exposed to x-ray film at 70°C.
The polymorphic markers tested included D18Mit55, D18Mit13, and
D13Mit13, which are syntenic to human 5q31 and D6Mit48, D5Mit40, and
D12Mit64, which are syntenic to genes within human 7q22-31.
Cytogenetic analysis and fluorescence in situ hybridization (FISH).
A trypsin-Giemsa banding technique was used to analyze cells from bone
marrow and spleen. Metaphase cells from short-term (24 to 72 hours)
unstimulated cultures were examined. Ten metaphase cells were examined
each from the bone marrow and spleen cultures for each mouse.
Chromosomes were identified using the standardized mouse karyotype as
described by Cowell.23 FISH was performed as described
previously.24 Briefly, a biotin-labeled Nf1 probe was prepared by nick-translation using Bio-16-dUTP (Enzo Diagnostics). Hybridization was detected with fluorescein-conjugated avidin (Vector
Laboratories), and chromosomes were identified by staining with
4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). Images were
obtained using a Zeiss Axiophot microscope coupled to a cooled charge
coupled device (CCD) camera. Separate images of DAPI-stained chromosomes and the hybridization signal were merged using image analysis software (NU200, Photometrics Inc, Phoenix, AZ and NIH Image
1.57, National Institutes of Health, Bethesda, MD). The Nf1 probe used for FISH was a 10.6 kb genomic lambda clone
containing exon 31 and flanking intron sequences.
| |
RESULTS |
Leukemia in Nf1+/+ and Nf1+/
mice.
Myeloid disorders developed in 4 of 101 Nf1+/+ mice, 2 of which
received CY (Table 1). In contrast, myeloid
malignancies were diagnosed in 14% of the untreated Nf1+/
mice (8 of 58), in 25% of the etoposide-treated animals (8 of 32), and
in 38% (14 of 37) of the mice assigned to the CY group (Table 1).
Kaplan-Meier plots comparing disease incidence over time in
Nf1+/+ and Nf1+/ mice that received no treatment,
etoposide, or CY are shown in Fig 1.
Nf1+/ mice that received either drug had a significantly higher rate of disease than wild-type animals treated in parallel (Fig
1). Treated and untreated Nf1+/ mice were also compared to
ascertain the relative contributions of Nf1 genotype and
chemotherapy exposure to leukemia susceptibility. This analysis showed
that the incidence of disease was significantly higher, and the latency period shorter, in the Nf1+/ mice that received CY (0.004 v untreated Nf1+/ mice by pairwise logrank
statistics), but not in the etoposide group (P = .2 v
the untreated group). The in vivo leukemogenic effect of CY was
restricted to Nf1+/ mice as Nf1+/+ animals in the
control and CY-treated groups had similar rates of leukemia (Table 1).
The incidence of leukemia was higher in Nf1+/ mice from the
inbred 129/Sv background than in 129/Sv × C57BL/6 animals (Table 1),
although these differences did not achieve statistical significance.
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| Fig 1.
Kaplan-Meier plots showing the proportion of
Nf1+/+ and Nf1+/ mice surviving without
leukemia. Nf1+/+ mice are shown as an unbroken line and
Nf1+/ mice as a dotted line. (A) Data from untreated mice;
(B) data from the etoposide group; (C) data from the CY group.
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A myeloproliferative phenotype was observed in most diseased mice that
was similar in control and chemotherapy-treated animals. This MPD was
characterized by elevated peripheral blood leukocyte counts with a high
percentage of mature neutrophils and monocytes (Fig
2). The mean WBC count was 31,000/µL
(range, 20,000 to 98,000), and the mean myeloid cell count was
28,000/µL (range, 14,000 to 88,000). Blood smears showed a variable
degree of myeloid differentiation with some containing greater than
80% mature neutrophils and others showing 30% to 40% monocytes and
monocytoid cells. Some smears showed rare blasts. Platelet counts and
hemoglobin values were normal in mice with MPD and immature erythroid
lineage cells were not seen in the peripheral blood. There was no
consistent relationship between treatment group, WBC count, and the
degree of myeloid maturation visible on blood smears. The bone marrows
of mice with MPD showed an overwhelming predominance of myeloid cells
with a shift toward immature elements, and sections of the spleen
showed expansion of red pulp with infiltration of myeloid cells at
various stages of differentiation admixed with areas of erythropoiesis (Fig 2). This MPD is similar to the JMML-like disorder that arises after adoptive transfer of Nf1/ fetal liver cells into
irradiated recipient mice.17 A disease phenotype more
consistent with acute leukemia was seen in one CY-treated
Nf1+/ mouse and in one mouse that received etoposide. Both
animals had WBC counts >150,000/µL with large numbers of blasts and
few mature neutrophils in the peripheral blood. The CY-treated mouse
also had anemia (hemoglobin level, 5.7 g/dL) and thrombocytopenia.
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| Fig 2.
Tissue sections from CY-treated Nf1+/ mice
with MPD. (A and B) Blood and bone marrow smears showing immature and
well differentiated myeloid cells. (C) A cytocentrifuge preparation of
spleen cells stained with the myeloid lineage marker nonspecific
esterase demonstrates many positive cells (brown stain). (D) A spleen
section shows a dense infiltrate of myeloid cells within the red pulp.
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Laboratory investigation of murine leukemias.
LOH at Nf1 correlated with clinical evidence of leukemia in
Nf1+/ mice (Table 2) and this
invariably involved loss of the wild-type Nf1 allele (Fig
3). Within the CY-treated group, leukemic cells from 129/Sv × C57BL/6 mice showed a much lower incidence of LOH
than cells from 129/Sv animals (Table 2). Both animals with evidence of
acute leukemia had LOH in hematopoietic tissues. In mice with MPD, LOH
was not consistently associated with higher leukocyte counts or with
increased numbers of immature myeloid cells. Unexpectedly, we detected
LOH at sacrifice in the hematopoietic tissues of 18% of mice that did
not fulfill the criteria used to diagnose leukemia. Most of these
animals appeared well and WBC counts <10,000/µL and absence of
prominent myeloid infiltrates in splenic sections. These results
implicate inactivation of Nf1 as an early event that confers an
in vivo proliferative advantage upon a clone of cells, but also suggest
that additional mutations are required to produce the characteristic
MPD. LOH in the absence of leukemia was much more common in 129/Sv mice
than in 129/Sv × C57BL/6 animals (Table 2). Among mice without
leukemia, LOH was relatively common in control animals but infrequent
in the etoposide-treated cohort (Table 2).
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| Fig 3.
Southern blot analysis of tissues from Nf1+/
mice with MPD. The 3.2-kb restriction fragment corresponds to the
targeted Nf1 allele, and the 2.4-kb band is derived from the
wild-type allele. DNA extracted from the bone marrows or spleens of
five mice with leukemia (L) show absence or a marked reduction in the
wild-type Nf1 allele compared to paired tail (T) DNA
specimens.
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Cytogenetic analysis of bone marrow and spleen cells from 6 mice with
MPD (5 CY-treated mice and 1 from the etoposide group) revealed a
normal karyotype (Fig 4A). To ascertain if
LOH on Southern blots was associated with submicroscopic deletions of
Nf1 or with duplication of the mutant allele, we used a genomic
Nf1 probe from the disrupted segment of the gene to perform
FISH analysis of hematopoietic cells from 3 of these 6 mice. FISH
showed 2 structural copies of the Nf1 gene in each case (Fig
4B). We also used six polymorphic microsatellite markers to examine
bone marrow DNA from mice with t-ML for LOH at loci syntenic to regions
of human chromosomes 5 and 7 that are frequently deleted in humans with t-MDS and t-AML, but found none (data not shown). Similarly, Southern blot analysis of specimens from etoposide-treated mice did not show
rearrangements of Mll when hybridized with a probe from the human MLL breakpoint cluster region that detects virtually all of the breakpoints in human leukemias (data not shown).
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| Fig 4.
(A) Cytogenetic analysis of spleen cells from a
CY-treated mouse with LOH at Nf1 shows a normal diploid
karyotype. (B) FISH of the same specimen shown in (A) with a genomic
Nf1 probe shows 1 copy of the gene on each chromosome 11 homolog.
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DISCUSSION |
This study establishes an in vivo model of therapy-induced myeloid
malgnancies in Nf1+/ mice that will facilitate basic and translational research studies of this important clinical disorder. In
human t-MDS/t-AML, frequent deletions involving chromosomes 5 and 7 have implicated loss of gene function in genotoxin-induced leukemogenesis. How alkylating agents actually cause leukemia is
unknown; however, CY increases the frequency of somatic inactivation of
target genes in a variety of assays.25 In contrast,
leukemias that arise after exposure to topoisomerase II inhibitors are
associated with recurring chromosomal translocations involving the
MLL gene that result in the production of dominantly acting
chimeric proteins. If the leukemias that develop after treatment with
alkylating agents predominately involves the inactivation of specific
target genes, it is possible that some human patients who develop t-ML after alkylator-based chemotherapeutic regimens are highly susceptible because of germline mutations of undiscovered tumor-suppressor genes
that, like NF1, restrain the growth of immature myeloid cells.
We used clinical criteria to diagnose leukemia because the bone marrows
of some children with NF1 who develop malignant myeloid disorders do
not show LOH at NF1.14,19,21 In this study, mice with clinical evidence of MPD or AML had a threefold higher rate of LOH
at Nf1 than mice without these findings. The presence of somatic LOH in hematopoietic tissues supports the clonal nature of
these myeloid disorders. MPDs with and without LOH had similar features
to the myeloid disorder that emerges after adoptive transfer of
Nf1/ fetal liver cells into irradiated recipient
mice.17 Together with the extraordinary increase in the
incidence of leukemia in Nf1+/ versus Nf1+/+
animals, these observations provide evidence that inactivation of
Nf1 is a central event in leukemogenesis even in the absence of
LOH. If this is true, it is likely the wild-type Nf1 allele is
inactivated in bone marrows without LOH by subtle somatic mutations. An
alternative consideration is that some Nf1+/ mice develop
myeloid malignancies through a genetic pathway that does not involve
biallelic inactivation of Nf1, as has recently been shown for a
subset of tumors from heterozygous p53 knockout
mice.26 Experiments using techniques that can identify point mutations will be required to distinguish between these possibilities. Nf1 is a very large gene, and protein truncation has proven to be the most efficient method for detecting subtle mutations in normal and leukemic cells from NF1
patients.20,27,28
Adoptive transfer of Nf1/ fetal liver cells into
irradiated mice consistently induces a MPD with features of
JMML.17 Inasmuch as these data suggested that inactivation
of Nf1 in early hematopoietic cells might be both necessary and
sufficient to induce clinical disease, we were surprised to detect LOH
at Nf1 in hematopoietic tissues from 18% of Nf1+/
mice with normal WBC counts. This idea that genetic alterations in
addition to inactivation of Nf1 are required for clinical
disease is consistent with the relatively long latency between CY
exposure and the onset of t-ML in Nf1+/ mice (Fig 1). Cooperating
somatic mutations such as bone marrow monosomy 7 and epigenetic events
have also been identified in human NF1-associated myeloid
disorders.21,29 It will be of interest to determine if LOH
can be detected in circulating blood cells some months before the onset
of leukocytosis and splenomegaly in Nf1+/ mice.
We did not inject bone marrow cells from Nf1+/ mice that
acquired myeloid disorders associated with LOH into secondary hosts. In
our hands, transferring marrow from recipients previously engrafted with Nf1/ fetal liver cells consistently induces MPD in
irradiated, but not in unirradiated, mice (data not shown). In an
interesting experiment, Largaespada et al17 crossed a
mutant Nf1 allele into the BXH2 line of mice in which a
leukemogenic retrovirus is transmitted vertically from mother to pups.
They observed preferential viral integration into the wild-type
Nf1 allele, shortened latency, and a change in disease
phenotype from MPD to AML.17 Their finding of other
somatically acquired leukemia-specific viral integrations within these
clones implicated alterations in addition to inactivation of
Nf1 in progression from MPD to AML. Adoptive transfer into secondary recipients provides a way of further characterizing therapy-induced myeloid disorders arising in Nf1 mice and may be especially informative in rare cases that show features of acute leukemia.
LOH at Nf1 and clinical leukemia were more common in homozygous
129/Sv mice than in 129/Sv × C57BL/6 animals. This was true in both
control and CY-treated mice. Thus, 129/Sv hematopoietic cells are
unexpectedly prone to spontaneously undergo LOH at Nf1 followed
by clonal expansion. Rates of cancer in the F1 progeny of crosses
between two inbred mouse strains often correlate poorly with parental
rates and may be higher, lower, or unchanged.30 The net
effect of our having assigned disproportionate numbers of 129/Sv × C57BL/6 mice to the CY group is to understate the magnitude of the
leukemogenic effect of this agent. CY-treated mice showed a higher
incidence of clinical leukemia than the control group irrespective of
genotype (58% v 17% in strain 129/Sv and 28% v 0%
in strain 129/Sv × C57BL/6; Table 1).
LOH was less frequent in CY-treated 129/Sv × C57BL/6 mice with MPD
than in any of the 129/Sv cohorts. This low incidence suggests that the
mechanism of Nf1 inactivation in 129/Sv × C57BL/6
hematopoietic cells involves subtle alkylator-induced mutations rather
than loss of the wild-type allele. Consistent with this, Shoemaker et
al31 recently identified somatic Apc point
mutations caused by transitions or transversions in intestinal tumors
from multiple intestinal neoplasia (Min) mice that had been exposed to
the alkylating agent N-ethyl-N-nitrosourea (ENU). Interestingly, other
tumors from this ENU-exposed cohort showed LOH at Apc. Taken
together with our data from CY-treated mice, these data suggest that
mechanisms of alkylator-induced tumor suppressor gene inactivation in
colonic and hematopoietic cells include somatic rearrangements that
result in LOH as well as subtle intragenic events.
Intestinal tumors that spontaneously arise in Min mice show LOH at
Apc with apparent loss of an entire chromosome 18 homolog.32 However, in a line of mice that carried
mutations of the Apc and Dpc4 tumor suppressor genes in
cis, intestinal tumorigenesis was associated with apparent loss
of one entire chromosome homolog with duplication of the mutant
chromosome.33 Consistent with this, FISH analysis of murine
leukemias with LOH showed an Nf1 allele on each chromosome 11 homolog. Although deletion of the chromosome containing the normal
tumor suppressor gene allele followed by duplication of the mutant
homolog has been proposed as a likely underlying
mechanism,33 other models are also plausible. Mitotic
nondisjunction resulting in two copies of the mutant homolog might
occur first, with subsequent loss of the normal chromosome. Alternatively, the DNA segment that contains the normal allele might be
replaced by a homologous segment from the mutant chromosome by a double
mitotic recombination event, as has been reported in a human
NF1-associated leukemia.34
Haran-Ghera et al35 previously observed a weak leukemogenic
effect of multiple doses of CY when this agent was administered with
radiation and dexamethasone to SJL/J mice, a strain that is susceptible
to radiation-induced AML. However, CY did not induce leukemia in the
absence of radiation, and only cooperated with radiation when it was
combined with dexamethasone.35 In contrast, we have
developed a murine model of t-ML based on clinical observations in NF1
patients21 in which CY alone efficiently induces myeloid leukemia in Nf1+/ mice.
Our data provide direct experimental evidence that exposure to a
commonly used cancer chemotherapeutic agent can cooperate with a
genetic predisposition in the development of myeloid malignancies. Although human patients with t-MDS/t-AML frequently show peripheral blood cytopenias when they seek medical attention, their bone marrows
are hypercellular and the disease typically evolves into a frankly
proliferative phase with time. Similarly, Nf1+/ mice only
exhibit overproliferation of myeloid cells months after exposure to CY.
As in humans, LOH in murine hematopoietic cells is associated with a
copy of the mutant Nf1 allele on each chromosomal homolog. The
relevance of this model to human leukemia is further suggested by the
presence of genetic alterations that deregulate Ras signaling in many
human myeloid leukemias,36,37 and by the finding of activating RAS mutations in the bone marrows of some patients with t-AML.38,39 Although our data implicating mutations of genes in addition to Nf1 in murine leukemogenesis are also
consistent with observations in human patients, we did not detect LOH
with polymorphic markers from regions of the murine genome that are syntenic to human 5q31 and 7q22. There are a number of potential explanations for these findings, including: (1) the probes used might
be some distance from the critical murine loci, (2) the relevant murine
genes may be inactivated by somatic mutations which do not result in
LOH, (3) loss of DNA sequences synteic to human 5q31 and 7q22 could be
late events in progression of MPD to AML that had not occurred by the
time of sacrifice, and/or (4) a different spectrum of cooperating genes
might be mutated in human and murine leukemias. The nature of the
alterations that are involved in alkylator-related leukemias awaits
identification of additional target genes in both species.
This novel model provides a rigorous in vivo system to address a number
of important (and in some cases controversial) issues in
therapy-related myeloid disorders including the relative leukemogenic potential of different alkylating agents, the role of dose intensity, and the additive effects (if any) of alkylating agents and external beam radiotherapy. Furthermore, molecular analysis at Nf1 may elucidate the mechanistic basis of genetic damage induced by specific alkylating agents in immature hematopoietic cells. Nf1+/
knockout mice will also be useful for testing the utility of surrogate markers of gene mutation such as inactivation of Hprt to
ascertain if exposure to specific mutagens portends an elevated risk of leukemia and to investigate chemopreventive strategies. Finally, these
results have implications for the care of individuals with NF1 who
develop neoplasms, because they suggest that alkylator-based regimens
should be avoided whenever possible.
| |
ACKNOWLEDGMENT |
We thank Peter Houghton for suggesting the chemotherapy drug schedules
used in this study, Rafael Espinosa III for assistance with FISH
analysis, Anthony Fernald for assistance with cytogenetic studies,
Nancy Zeleznik-Le and Janet Rowley for the MLL probe, Alexander
Muller and Arnold Levine for information about polymorphic loci in
129/Sv × C57BL/6 hybrid mice, Sandra-Luna Fineman and Nancy Swigonski
for assistance with statistical analyses, and Ben Yen and Ivy Hseih for
preparing sections of murine tissues.
| |
FOOTNOTES |
Submitted December 22, 1998; accepted March 25, 1999.
N.M. and B.R.T. contributed equally to this work.
Supported by American Cancer Society Research Grant No. DB-80030 and by
National Institutes of Health (NIH) Grants No. R01 CA72614 and P01
CA40046. N.M. was supported by NIH Training Grant No. DK07636. T.J. is
an Investigator of the Howard Hughes Medical Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kevin M. Shannon, MD, Department of
Pediatrics, University of California, Room HSE-302, Box 0519; San
Francisco, CA 94143-0519; e-mail: kevins{at}itsa.ucsf.edu.
| |
REFERENCES |
1.
Rowley JD, Golomb HM, Vardiman J:
Nonrandom chromosomal abnormalities in acute nonlymphocytic leukemia in patients treated for Hodgkin disease and non-Hodgkin lymphoma.
Blood
50:759, 1977
2.
Le Beau M, Albain KS, Larson RA, Vardiman J, Davis E, Blough R, Golomb H, Rowley J:
Clinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia: Further evidence for characteristic abnormalties of chromosomes 5 and 7.
J Clin Oncol
3:325, 1986
3.
Pedersen-Bjergaard J, Phillip P:
Cytogenetic characteristics of therapy-related acute nonlymphocytic leukemia, preleukemia, and acute myeloproliferative syndrome: Correlation with clinical data in 61 consecutive cases.
Br J Hematol
66:199, 1987[Medline]
[Order article via Infotrieve]
4.
Pedersen-Bjergaard J, Rowley JD:
The balanced and unbalanced chromosomal aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation.
Blood
83:2780, 1994[Abstract/Free Full Text]
5.
Ratain MJ, Kaminer LS, Bitran JD, Larson RA, Le Beau MM, Skosey C, Rowley JD:
Acute nonlymphoblastic leukemia following etoposide and cisplatin combination chemotherapy for advanced small cell carcinoma of the lung.
Blood
70:1212, 1987
6.
Levine E, Bloomfeld C:
Leukemias and myelodysplastic syndromes secondary to drug, radiation, and environmental exposure.
Semin Oncol
19:47, 1992[Medline]
[Order article via Infotrieve]
7.
Pui C-H, Behm FG, Raimondi SC, Dodge RK, George SL, Rivera GK, Mirro JJ, Kalwinsky DK, Dahl GV, Murphy SB, Crist WM, Williams DL:
Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia.
N Engl J Med
321:136, 1989[Abstract]
8.
Smith MA, Rubinstein L, Ungerleider RS:
Therapy-related acute myeloid leukemia following treatment with epipodophyllotoxins: Estimating the risks.
Med Pediatr Oncol
23:86, 1994[Medline]
[Order article via Infotrieve]
9.
Riccardi VM, Eichner JE:
Neurofibromatosis. Baltimore, MD, Johns Hopkins University Press, 1986.
10.
Bader JL, Miller RW:
Neurofibromatosis and childhood leukemia.
J Pediatr
92:925, 1978[Medline]
[Order article via Infotrieve]
11.
Stiller CA, Chessells JM, Fitchett M:
Neurofibromatosis and childhood leukemia/lymphoma: A population-based UKCCSG study.
Br J Cancer
70:969, 1994[Medline]
[Order article via Infotrieve]
12.
Arico M, Biondi A, Pui C-H:
Juvenile myelomonocytic leukemia.
Blood
90:479, 1997[Free Full Text]
13.
Boguski M, McCormick F:
Proteins regulating Ras and its relatives.
Nature
366:643, 1993[Medline]
[Order article via Infotrieve]
14.
Shannon KM, O'Connell P, Martin GA, Paderanga D, Olson K, Dinndorf P, McCormick F:
Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders.
N Engl J Med
330:597, 1994[Abstract/Free Full Text]
15.
Kalra R, Paderanga D, Olson K, Shannon KM:
Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras.
Blood
84:3435, 1994[Abstract/Free Full Text]
16.
Jacks T, Shih S, Schmitt EM, Bronson RT, Bernards A, Weinberg RA:
Tumorigenic and developmental consequences of a targeted Nf1 mutation in the mouse.
Nature Genet
7:353, 1994[Medline]
[Order article via Infotrieve]
17.
Largaespada DA, Brannan CI, Jenkins NA, Copeland NG:
Nf1 deficiency causes Ras-mediated granulocyte-macrophage colony stimulating factor hypersensitivity and chronic myeloid leukemia.
Nat Genet
12:137, 1996[Medline]
[Order article via Infotrieve]
18.
Bollag G, Clapp DW, Shih S, Adler F, Zhang Y, Thompson P, Lange BJ, Freedman MH, McCormick F, Jacks T, Shannon K:
Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in murine and human hematopoietic cells.
Nat Genet
12:144, 1996[Medline]
[Order article via Infotrieve]
19.
Miles DK, Freedman MH, Stephens K, Pallavicini M, Sievers E, Weaver M, Grunberger T, Thompson P, Shannon KM:
Patterns of hematopoietic lineage involvement in children with neurofibromatosis, type 1, and malignant myeloid disorders.
Blood
88:4314, 1996[Abstract/Free Full Text]
20.
Side L, Taylor B, Cayouette M, Connor E, Thompson P, Luce M, Shannon K:
Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders.
N Engl J Med
336:1713, 1997[Abstract/Free Full Text]
21.
Maris JM, Wiersma SR, Mahgoub N, Thompson P, Geyer RJ, Lange BJ, Shannon KM:
Monosomy 7 myelodysplastic syndrome and other second malignant neoplasms in children with neurofibromatosis type 1.
Cancer
79:1438, 1997[Medline]
[Order article via Infotrieve]
22.
Shannon KM, Turhan AG, Chang SSY, Bowcock AM, Rogers PCJ, Carroll WL, Cowan MJ, Glader BE, Eaves CJ, Eaves AC, Kan YW:
Familial bone marrow monosomy 7: Evidence that the predisposing locus is not on the long arm of chromosome 7.
J Clin Invest
84:984, 1989
23.
Cowell JK:
A photographic representation of the variability in the G-banded structures of the chromosome in the mouse karyotype.
Chromosoma
89:294, 1984[Medline]
[Order article via Infotrieve]
24.
Rowley JD, Diaz MO, Espinosa R, Patel YD, van Melle E, Ziemin S, Le Beau MM:
Mapping chromosome band 11q23 in acute leukemia with biotinylated probes: Identification of 11q23 breakpoints within a yeast artifical chromosome.
Proc Natl Acad Sci USA
87:9358, 1990[Abstract/Free Full Text]
25.
Anderson D, Bishop JB, Garner RC, Ostrosky-Wegman P, Selby PB:
Cyclophosphamide: Review of its mutagenicity for an assessment of potential germ cell risks.
Mutation Res
330:115, 1995
26.
Venkatachalam S, Shi YP, Jones SN, Vogel H, Bradley A, Pinkel D, Donehower LA:
Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.
EMBO J
17:4657, 1998[Medline]
[Order article via Infotrieve]
27.
Heim R, Silverman L, Farber R, Kam-Morgan L, Luce M:
Screening for truncated NF1 proteins.
Nat Genet
8:218, 1994[Medline]
[Order article via Infotrieve]
28.
Heim R, Kam-Morgan L, Binnie C, Corns D, Cayouette M, Farber R, Aylsworth A, Silverman L, Luce M:
Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.
Human Mol Genet
4:975, 1995[Abstract/Free Full Text]
29.
Shannon KM, Watterson J, Johnson P, O'Connell P, Lange B, Shah N, Kan YW, Priest JR:
Monosomy 7 myeloproliferative disease in children with neurofibromatosis, type 1: Epidemiology and molecular analysis.
Blood
79:1311, 1992[Abstract/Free Full Text]
30.
Smith GS, Walford RL, Mickey MR:
Lifespan and incidence of cancer in selected long-lived inbred mice and their F1 hybrids.
J Natl Cancer Inst
50:1195, 1973
31.
Shoemaker AR, Luongo C, Moser AR, Marton LJ, Dove WF:
Somatic mutational mechanisms involved in intestinal tumor formation in Min mice.
Cancer Res
57:1999, 1997[Abstract/Free Full Text]
32.
Luongo C, Moser AR, Gledhill S, Dove WF:
Loss of Apc+ in intestinal adenomas from Min mice.
Cancer Res
54:5847, 1994
33.
Takaku T, Oshima M, Miyoshi M, Matsui M, Seldin MF, Taketo M:
Intestinal tumorigenesis in compound mutant mice of both Dpc4 and Apc genes.
Cell
92:645, 1998[Medline]
[Order article via Infotrieve]
34.
Stephens K, Weaver M, Leppig K, Side LE, Shannon KM, Maruyama K:
Somatic confined uniparental disomy of the NF1 gene region in myeloid leuekmic cells of an NF1 patient mimics a loss of hetrozygosity due to deletion (abstract).
Am J Human Genet
59:A5, 1996 (suppl)
35.
Haran-Ghera N, Peled A, Krautghamer R, Resnitzkt P:
Initiation and promotion of radiation-induced myeloid leukemia.
Leukemia
6:689, 1992[Medline]
[Order article via Infotrieve]
36.
Sawyers CL, Denny CT:
Chronic myelomonocytic leukemia: tel-a-kinase what ets all about.
Cell
77:171, 1994[Medline]
[Order article via Infotrieve]
37.
Shannon KM:
The Ras signaling pathway and the molecular basis of myeloid leukemogenesis.
Curr Opin Hematol
3:305, 1995
38.
Pedersen-Bjergaard J, Janssen JWG, Lyons J:
Point mutations of the ras protooncogenes and chromosome aberrations in acute nonlymphocytic leukemia and preleukemia related to therapy with alkylating agents.
Cancer Res
48:1812, 1988[Abstract/Free Full Text]
39.
Side LE, Teel K, Wang P, Mahgoub N, Larson R, Le Beau MM, Shannon KM:
Activating RAS mutations in therapy-related myeloid disorders associated with deletions of chromosomes 5 and 7 (abstract).
Blood
88:566a, 1996 (abstr, suppl 1)
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