Blood, 15 March 2001, Vol. 97, No. 6, pp. 1882-1884
BRIEF REPORT
Time course of increasing numbers of mutations in the granulocyte
colony-stimulating factor receptor gene in a patient with
congenital neutropenia who developed leukemia
Christoph A. Tschan,
Christina Pilz,
Cornelia Zeidler,
Karl Welte, and
Manuela Germeshausen
From the Department of Paediatric Haematology and
Oncology, Hannover Medical School Hannover, Germany.
 |
Abstract |
Point mutations in the granulocyte colony-stimulating factor
receptor (G-CSFR) gene have been linked to the
development of secondary leukemia in patients with congenital
neutropenia (CN). This report presents data on a now 18-year-old
patient with CN who has received G-CSF treatment since 1989 and who
developed acute myeloid leukemia (AML) in 1998. To evaluate whether
there is an association between the occurrence of point mutations of the G-CSFR gene and development of secondary AML,
DNA/messenger RNA of neutrophils and mononuclear cells from this
patient were analyzed at different time points by polymerase chain
reaction and subsequent cloning by DNA sequencing of representative
numbers of individual clones. Findings suggest an increasing
instability of the G-CSFR gene in time as judged by
increasing numbers of mutations proposed to be one important step in
the development of AML in this patient.
(Blood. 2001;97:1882-1884)
© 2001 by The American Society of Hematology.
| |
Introduction |
We describe the time course of a stepwise
acquisition of different granulocyte colony-stimulating factor receptor
(G-CSFR) gene mutations in a patient with severe congenital
neutropenia (CN, Kostmann syndrome). In this patient, the occurrence of
these mutations significantly correlated with the transformation of CN
into acute myeloid leukemia (AML).
Congenital neutropenia is characterized by a maturation arrest of
myeloid progenitor cells at the promyelocyte-myelocyte stage, resulting
in absence or low levels of mature neutrophils. Patients with CN
usually develop recurrent localized or systemic bacterial and fungal
infections, beginning in early infancy. CN treatment with granulocyte
colony-stimulating factor (G-CSF) results in an increase of absolute
neutrophil count (ANC), significantly fewer infections, and improvement
of quality of life.1
| |
Study design |
We report data on an 18-year-old girl who was diagnosed with CN
in 1982, at the age of 7 months. In 1989, the patient presented with a
history of severe bacterial infections and an ANC persistently below
0.2 × 109/L. G-CSF treatment was initiated with 3 µg/kg per day subcutaneously and her ANC increased to above
1.0 × 109/L. During the following 9 years of G-CSF
treatment, the patient developed normally and did clinically well
without severe infections.
At a routine hospital visit in March 1998, her white blood count was
5.1 × 109/L and contained 11% myeloid blast cells.
Acute myeloid leukemia (AML-M5b) was diagnosed by morphologic criteria.
One third of the leukemic cells demonstrated monosomy 7 and two thirds
showed a tetraploid karyotype. Treatment was initiated according to the AML-BFM-98 protocol,2 followed by bone marrow
transplantation (BMT). Bone marrow was donated by her haplo-identical
(2 loci mismatched) half-brother in July 1998.
| |
Results and discussion |
Patients with CN have an increased risk of approximately 10% to
develop myelodysplastic syndrome (MDS) or AML or
both.1,3,4 Acquired nonsense mutations in a critical
region between nucleotide (nt) 2360 and nt 2430 of the
G-CSFR gene encoding an intracellular part of the G-CSFR
protein, which contributes to myeloid proliferation and maturation
signaling, are thought to be involved in the leukemogenesis in these
patients.3,5,6 These G-CSFR gene
mutations introduce a stop codon, potentially leading to a loss of
parts of the carboxyterminal domain of the receptor protein (Figure
1). Interestingly, receptor proteins
truncated in this way were shown to increase cell proliferation with
arrest of differentiation.5
View larger version (24K):
[in this window]
[in a new window]
| Figure 1.
Sequence of acquisition of mutations in the CN patient
demonstrated on a schematic presentation of the G-CSFR.
Arrows indicate positions of acquired point mutations of the G-CSFR
mRNA at nt 2363, 2390, and 2414. The mutations either lead to an amino
acid exchange from aspartate (asp) to asparagine (asn; nt 2363) or from
glutamine (gln) to stop codons (TAA and TAG; nt 2390 and 2414). Gray
boxes indicate the homologous domains (box 1-3) conserved in
the cytokine receptor family. EC indicates extracellular; TM,
transmembrane; IC, intracellular domain; compl, complete; aa, amino
acids; nt, nucleotide.
|
|
To evaluate a potential association between point mutations in
the critical region of the G-CSFR gene and secondary AML, we examined DNA of bone marrow or messenger RNA (mRNA) of
polymorphonuclear cells (PMCs) and mononuclear cells (MNCs) of our
patient. Samples from different time points were analyzed by polymerase
chain reaction (PCR) amplification of a region between nt 2306 and 2906 and subsequent cloning followed by DNA sequencing of representative
numbers of individual Escherichia coli clones (Table
1; nucleotide numbering refers to
Fukunaga and colleagues7). Analyses of genomic DNA (gDNA)
from 1989, a time point before initiation of G-CSF therapy, revealed a
point mutation at nt 2363 leading to an exchange from aspartate to
asparagine. Up to now this mutation could only be shown in myeloid
cells of 2 other CN patients (M.G., unpublished results, January
2001). None of the tested normal donors or patients with other
leukemic or preleukemic disorders demonstrated this point mutation. We
detected an additional G-CSFR gene mutation at nt 2414 leading to a stop codon and to a truncated G-CSFR protein in samples
from May 1994 (Figure 1). At this time, clonal heterogeneity was
confirmed by detection of clones harboring either the mutation at nt
2363 or 2414 alone, or a combination of both mutations. Samples of MNC
RNA from April 1996 revealed similar results (Table 1). The sequence of
acquisition of mutations, in particular at that stage, when the second
mutation (C2414T) is present in clones with as well as without the nt
2363 mutation (May 1994, April 1996) suggests that the nt 2414 mutation
occurred in different hematopoietic cell clones (with and without the
nt 2363 mutation). Another possibility could be that the nt 2414 mutation occurred in the nt 2363 clone, but more or less simultaneously
on the wild-type allele and the mutated allele.
View this table:
[in this window]
[in a new window]
|
Table 1.
Sequence analyses of different clinical stages in a
patient with congenital neutropenia who developed acute myeloid
leukemia
|
|
In 1997, karyotype analysis for the patient reported here was still
normal. At the time leukemia was diagnosed, 4 distinct variants of
mutated G-CSFR genes were detectable: one variant with the
mutation at nt 2363 alone, one with the mutation at nt 2414 alone, one
with both mutations, and another with a newly diagnosed nonsense
mutation at nt 2390, also introducing a stop codon (Figure 1 and Table1).
After induction of chemotherapy, the clones of leukemic cells
expressing mutations at nt 2414 and nt 2390 and displaying monosomy 7 disappeared. However, cells harboring the mutation at nt 2363 were still detectable during clinical remission until BMT was performed
in July 1998. After BMT, none of the mutations could be detected in the
patient's hematopoietic cells (Table 1).
The increasing number of mutations reported here document the
instability of the G-CSFR gene, which is proposed to be one of the reasons for malignant transformation.
Until now we detected 7 CN patients with multiple and 11 with
single nonsense mutations in the critical region of the
G-CSFR gene.3 To date, 5 patients with multiple
mutations and 6 patients with single mutations developed secondary
leukemia. Accordingly, we found a highly significant correlation
between the occurrence of one or more G-CSFR gene mutations
and leukemogenesis in CN patients rather than a correlation between the
number of different mutations and the risk of developing leukemia.
Furthermore, as CN patients with a single G-CSFR gene
mutation as well as CN patients with multiple mutations developed
secondary AML with a comparable incidence, multiple mutations may
indicate a general and possibly increasing instability of the
G-CSFR gene. The risk of developing AML in patients with CN
seems to increase drastically with the occurrence of the first single
mutation in the unstable G-CSFR gene as one early step in
leukemogenesis. Other chromosomal aberrations are rather late events
only detectable in the overt leukemic cells. Our data confirm that
mutations of the G-CSFR gene are acquired during the course
of disease and represent important initial steps from CN toward leukemia.
The contribution of the G-CSF therapy to the development of
G-CSFR gene mutations or leukemia remains unclear. However,
data from the pre-G-CSF era already demonstrate that CN is a
preleukemic disorder leading to leukemia in some patients. In addition,
data of the Severe Chronic Neutropenia International Registry
demonstrate that none of the patients with cyclic and idiopathic
neutropenia who were also treated with G-CSF with similar dosages and
for the same length of time developed either point mutations in the critical region of the G-CSFR gene or
leukemia.1,4
Taking into consideration the time course of mutations in the CN
patient reported here and the highly significant correlation between
the occurrence of G-CSFR gene mutations and leukemogenesis in CN patients, we recommend a yearly evaluation of G-CSFR
gene mutations in all CN patients to consider BMT options early if nonsense mutations in the critical region of the G-CSFR gene
are identified.1,3,4,6
| |
Footnotes |
Submitted September 5, 2000; accepted November 13, 2000.
Supported by a grant from the Deutsche Krebshilfe
(10-1548-We2).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Karl Welte, Medizinische Hochschule
Hannover, Paediatrische Haematologie und Onkologie,
Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; e-mail:
welte.karl{at}mh-hannover.de.
| |
References |
1.
Welte K, Boxer LA.
Severe chronic neutropenia: pathophysiology and therapy.
Semin Hematol.
1997;34:267-278[Medline]
[Order article via Infotrieve].
2. Creutzig U, Ritter J, Zimmermann M, Hermann J, Gadner H, for the
AML-BFM Study Group. Risk-adapted therapy and randomization in children
with AML: preliminary results of study AML-BFM 93. Haematol Blood
Transfus. 2000. In press.
3.
Germeshausen M, Tidow N, Pilz C, Tschan C, Zeidler C, Welte K.
G-CSF receptor mutations in patients with congenital neutropenia: frequency and implications in leukemic development [abstract].
Blood.
1999;94:45a.
4.
Freedman MH, Bonilla MA, Fier C, et al.
Myelodysplasia syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy.
Blood.
2000;96:429-436[Abstract/Free Full Text].
5.
Dong F, Brynes RK, Tidow N, Welte K, Lowenberg B, Touw IP.
Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia.
N Engl J Med.
1995;333:487-493[Abstract/Free Full Text].
6.
Tidow N, Pilz C, Teichmann B, et al.
Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.
Blood.
1997;89:2369-2375[Abstract/Free Full Text].
7.
Fukunaga R, Seto Y, Mizushima S, Nagata S.
Three different mRNAs encoding human granulocyte colony-stimulating factor receptor.
Proc Natl Acad Sci U S A.
1990;87:8702-8706[Abstract/Free Full Text].
Top
Abstract
Introduction
