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p53 Mutation as the Second Event in Juvenile Chronic
Myelogenous Leukemia in a Patient with
Neurofibromatosis Type 1
Drorit Luria, M.Sc.1
Smadar Avigad, Ph.D.1,2
Ian J. Cohen, M.B., Ch.B.2
Batia Stark, M.D.2
Raphael Weitz, M.D.3
Rina Zaizov, M.D.1,2
BACKGROUND. Young patients with neurofibromatosis type 1 (NF1) are at increased
risk of developing various malignancies, most of which are myeloid disorders. The
observed loss of NF1 allele in the myeloid malignancies of NF1 patients suggests
Cancer Molecular Genetics, Felsenstein Medical Research Center, Tel Aviv University, Tel
Aviv, Israel.
The Center for Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tiqwa, Israel.
The Center for Pediatric Neurology, Schneider
Children’s Medical Center of Israel, Petah Tiqwa,
Israel; Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
a role of NF1 as a tumor suppressor gene. Loss of 17p was found to be quite
frequent in neural crest tumors from patients with NF1, raising the possibility of
p53 tumor suppressor gene involvement in other NF1-related tumors.
METHODS. The authors studied mutations in the NF1 and p53 genes, using loss
of heterozygosity, single strand conformation polymorphism, heteroduplex and
sequencing analyses.
RESULTS. An NF1 germline mutation was identified in exon 31 of a child who
developed juvenile chronic myelogenous leukemia (JCML). The mutation was segregated within the proband’s family. A 14bp deletion at exon 6 of the p53 gene
was observed when JCML was diagnosed, and the wild-type p53 allele was lost
during progression of the disease. No loss of the normal NF1 allele could be
CONCLUSIONS. A germline mutation in the NF1 gene and sequential inactivation of
p53 alleles in the malignant clone of JCML raise the possibility of a correlation between
NF1 and p53 genes in the tumorigenesis of JCML. Cancer 1997;80:2013–8.
q 1997 American Cancer Society.
KEYWORDS: neurofibromatosis type 1, p53, juvenile chronic myelogenous leukemia,
Presented as a poster at the 27th SIOP meeting,
October 1995.
Supported by the Josefina Maus and Gabriela
Cesarman Maus Chair for Pediatric Hematology
This work is a partial fulfillment of the requirements for the Ph.D. degree of Drorit Luria at
the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
The authors thank Dr. M. Israel for reviewing
this manuscript and for his fruitful discussion
and suggestions.
Address for reprints: Smadar Avigad, Ph.D., the
Center for Pediatric Hematology Oncology,
Schneider Children’s Medical Center of Israel,
Petah Tiqwa, 49202, Israel.
Received March 7, 1997; revision received May
23, 1997; accepted May 23, 1997.
eurofibromatosis type 1 (NF1) is the most common autosomal
dominant disorder, affecting 1 in 3500 individuals. Major clinical
features are café-au-lait spots, benign neurofibromas, and Lisch nodules of the iris. Patients with NF1 are predisposed to certain types of
cancer that arise primarily in cells derived from the embryonic neural
crest.1 – 3 Young patients are at an increased relative risk of malignant
myeloid disorders, such as juvenile chronic myelogenous leukemia
(JCML), acute myeloid leukemia (AML), and monosomy 7 syndrome.3,4
The NF1 gene has been mapped to 17q11.2 and spans more than
350 kilobases (Kb) of genomic DNA; it encodes a 12 – 13 Kb messenger
RNA (mRNA) that consists of 59 exons.5 – 7
Neurofibromin, the protein encoded by the NF1 gene, includes
a domain that is homologous with yeast and mammalian GTPase
activating proteins (GAPs). It binds to ras with high affinity and accelerates the intrinsic GTPase activity of p21 ras proteins.8 Loss of the
normal NF1 allele in the neurofibrosarcomas and other tumors of
NF1 patients correlates with elevated levels of GTP-ras.9 – 12 These ob-
q 1997 American Cancer Society
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CANCER November 15, 1997 / Volume 80 / Number 10
Heteroduplex analysis of exon 31 in the NF1 gene in the
proband and his family is shown. The abnormal band was detected in the
proband and his father and sisters.
servations suggest that the NF1 gene functions as a
tumor suppressor gene.
Heterozygous mice (NF1//0) resemble NF1 patients in their high predisposition to pheochromocytomas and myeloid leukemia. The wild-type NF1 allele
is lost in approximately half the tumors from these
animals.13 In analysis of malignant bone marrow samples from children with NF1 and myeloid disorders,
heterozygosity was retained in 50% (9 of 18).11,14 The
same phenomenon was described in different tumors
that developed in patients with NF1.15 – 17 Moreover, in
these tumors, loss of the p53 wild-type allele occurred
quite frequently.15 – 18
Involvement of the p53 gene is not frequent in
AML, though p53 point mutations are noted, mainly
in advanced variants of myelodysplastic syndrome
(MDS) and AML, with a frequency of about 5 – 10%.19,20
A child with myeloproliferative disorder (MPD)
was referred to our center and was diagnosed as having NF1. We have identified a nonsense germline NF1
mutation in exon 31 that segregated in the affected
members of the family. A 14 bp deletion in exon 6 of
the p53 gene was identified when JCML was diagnosed
in the proband. No loss of heterozygosity (LOH) of the
wild-type NF1 allele could be identified, whereas LOH
of the p53 gene was observed in the disease progression to AML.
Case Report
A boy age 5.5 years was referred to our department in
December 1986 for evaluation of MPD. His father and
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paternal grandfather had NF1 with dermal neurofibromas, and three sisters had café-au-lait spots and
learning disabilities. The patient had had recurrent
respiratory infections and failure to thrive since infancy. Clinical examination revealed multiple café-aulait spots, facial papulonodular rash, prominent submandibular lymphadenopathy, and marked hepatosplenomegaly. Laboratory data revealed that hemoglobin was 7.8 g/dL, platelets 28,000/mL, and leukocyte
count 35,300/mL, with 72% neutrophils, 9% bands, 6%
monocytes, 5% blasts, and 9% lymphocytes. Fetal hemoglobin was 3.1% and LAP score was 84 (control,
260). Bone marrow (BM) aspiration showed hypercellularity with megaloblastic changes, myelomonocytic
hyperplasia with shift to the left, and basophilia of 1%
and 4% blasts. A decreased number of megakaryocytes
was noted. Natural killer activity in peripheral blood
was lower than in the control (7% vs. 30%). Studies of
hemopoiesis in BM and peripheral blood utilizing the
methylcellulose method revealed striking growth of
spontaneous colony forming unit-granulocytes monocytes ú300/105 cells plated (control, 7/105) and reduced growth of burst forming unit-erythroid 24/105
cells plated (control, 84/105). These data were compatible with JCML.21 Karyotype of BM cells revealed 47,XY
/21/46,XY. Trisomy 21 was detected only in the leukemic cells.
During the next 2 years, the patient continued to
have recurrent infections. Leukocyte counts increased,
platelet counts gradually decreased, and he developed
a tendency toward bleeding that persisted thereafter.
A treatment protocol for AML was initiated in March
1989 with temporary partial response. Two and a half
years later, acute myelomonocytic leukemia developed. The patient died in March 1992.
DNA extraction
DNA was extracted from peripheral blood cells (PBCs)
of the proband, his parents, and four sisters (informed
consent was obtained from the parents). DNA from the
BM was extracted only from the proband at different
stages of the myeloproliferative disorder. Extractions
were performed with lysis buffer, sodium dodecyl sulfate (SDS), and proteinase K, followed by the salting
out method for PBC and BM.22
Screening for mutations in the NF1 gene
Screening was performed using single strand conformation polymorphism (SSCP) and heteroduplex analyses. For SSCP, 2 – 3 ml of polymerase chain reaction
(PCR) product was mixed with 0.1% SDS and 10 mM
ethylenediamine tetraacetic acid, heat denatured, run
on 6% polyacrylamide gel at 400 V for 3 – 4 hours at 4
7C, and silver stained.23 For heteroduplex analysis, 10%
W: Cancer
p53 Mutation in an NF1 Patient/Luria et al.
Sequence analysis of
exon 31 in the NF1 gene is shown. A
transition of CAA to TAA in a heterozygous form, as detected in the peripheral blood cells (PBCs) of the proband,
is compared with the PBCs of his
mother (which were normal).
Hematologic Status in Correlation with Molecular and Cytogenetic Changes
% Blasts
tri 21
tri 21
tri 21
tri 21, 017p
WBC: white blood cell count; PLT: platelet count; Hb: hemoglobin; BM: bone marrow; NF1: neurofibromatosis type 1; M: mutation; L: loss of heterozygosity; tri 21: trisomy 21; 017p: deletion of the short arm of
chromosome 17.
of the PCR product was heat denatured, quenched for
2 minutes on ice, and reannealed at room temperature
for 3 – 7 hours. Samples were run on MDE gel (Hydrolink) for 13 – 14 hours at 110 V and stained with
ethidium bromide.
Sequencing was done by solid phase sequence analysis
using streptavidin 280 (Dynals) and the dideoxy
method of Sanger et al. (Sequenase 2.0, USB).24 The
primers used for sequence analysis of NF1 exons were
the same as those used for PCR.6
LOH analysis
LOH analysis was performed using microsatellites
within and flanking the NF1 gene.11,25 – 28 PCR products
were electrophoresed on 10% polyacrylamide gels at
100 V for 12 – 14 hours and stained with ethidium bromide.
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Analysis of the p53 gene
LOH analysis for the p53 gene was performed using a
VNTR region in intron 129 and restriction fragment
length polymorphisms (RFLPs) in exon 430 and intron
6.31 For sequence analysis, exons 5 – 9 were amplified
by PCR using the primers described.32
Heteroduplex analysis of exon 31 revealed a suspected heteroduplex fragment in the proband, his
father, and two of the affected sisters (Fig. 1). Sequence analysis of exon 31 revealed a C- to T-transition at codon 1972 (Glu-CAA), which created a stop
codon (TAA) in all affected members (Fig. 2) and
in the malignant cells of the proband. In the LOH
analysis, we compared the DNA from PBCs and BM
overloaded with 99% blasts dated from March 1992.
The DNA was informative for all the microsatellites
used, and heterozygosity was retained for the NF1
gene (data not shown).
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CANCER November 15, 1997 / Volume 80 / Number 10
Loss of heterozygosity (LOH) analysis of the p53 gene is
shown. LOH was identified in Case 1, our proband, using restriction fragment length polymorphism in exon 4 of the p53 gene. The polymerase
chain reaction product was digested with BstUI, resulting in two alleles:
259 bp and 160 / 99 bp. In the tumor (T), the 259 bp allele was lost.
Regarding p53 involvement, LOH of the p53 allele was detected in the sample from the AML stage
in two informative polymorphic sites, RFLP in exon
4 (Fig. 3) and in intron 6 (data not shown). Genomic
sequence analysis of exons 5 – 8 of the p53 gene revealed a deletion of 14bp in exon 6 creating a stop
codon (TGA at codon 223, Fig. 4). This mutation
was also identified at diagnosis using a special
primer that flanks the deletion identified in exon 6,
amplifying the mutant allele. Using another special
primer for intron 6, we were able to amplify mostly
blast cells; in this manner, we could study the LOH
in crucial BM samples from this child (Table 1),
even those with a low percentage of blasts. LOH
was identified in Sample 3 from 1988 and confirmed
by laser densitometer using PDI software (Dinco &
A boy with JCML was diagnosed as having familial
NF1. A mutation in exon 31 of the NF1 gene, a transition of C to T in codon 1972, was identified. This mutation created a stop codon that segregated with the NF1
phenotype of the proband, his father, and three sisters.
After diagnosis of JCML at age 5.5 years, the patient’s
clinical status gradually deteriorated with the development of AML, from which he died.
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FIGURE 4. Sequence analysis of exon 6 of the p53 gene is shown. A
14bp deletion created a shift in the tumor DNA sequence.
Children with NF1 are at increased risk of leukemia, particularly myelodysplastic disorders.33,34 The
BM of patients with myeloid leukemia carry ras mutations in 20 – 30% of cases.35,36 Leukemic cells of children with NF1 never show activating ras mutations
and, alternatively, delete the wild-type NF1 allele.11,35
In our proband, we were unable to identify LOH of
the wild-type NF1 allele using six informative microsatellites. Genetic evidence proves that NF1 functions
as a tumor suppressor gene in myeloid cells by negatively regulating the ras oncogene through its GAP domain.11 – 13 Yet, in 50% of myeloid disorders, including
tumors in heterozygous mice, no LOH of the normal
NF1 allele could be identified. Moreover, the identified
germline mutation in exon 31 is downstream to the
GAP domain. This suggests that neurofibromin might
affect the ras oncogene in other ways,37 or, alternatively, that ras is not always involved in myeloid disor-
W: Cancer
p53 Mutation in an NF1 Patient/Luria et al.
ders of patients with NF1. Neurofibromin and activated ras may contribute differently to the negative
control of neural crest cells, as shown in different cell
lines derived from patients with NF1.9,10,38 – 40
To determine possible additional genetic alterations in the pathogenesis of the malignant myeloid
disorder, and in view of several publications on the
role the p53 gene in the oncogenesis of NF1 tumors,15–17
we analyzed p53 involvement in the proband. A mutation in exon 6, a 14 bp deletion leading to a premature
stop codon, was identified when JCML was diagnosed.
It was important to determine whether the p53 gene
was present when the diagnosis was made. Using a
specific primer for the mutant allele, we identified the
deletion in p53 exon 6 already at diagnosis. LOH of the
wild-type p53 gene was identified in the progression
of the disease to AML and confirmed by cytogenetic
analysis (Table 1). LOH of 17p has been found to be
frequent in tumors of neural crest origin in NF1 patients (occurring in 65%), suggesting the involvement
of the p53 suppressor gene. Mutations in the p53 gene
were identified in 7 of 12 NF1 tumors screened
(58%).15 – 18 At this stage, we cannot rule out the possibility of an additional mutation in the NF1 gene, as a
second hit to the germline mutation contributing to
the leukemogenesis in the patient with NF1 whom we
have described. The inactivation of p53 alleles might
fit into the multistep model for childhood MPD proposed by Butcher et al.41
Our results suggest that heterozygous NF1 mutation combined with sequential inactivation of p53 resulted in the tumorigenesis of JCML in a child with
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