Blood, 1 February 2001, Vol. 97, No. 3, pp. 829-830
CORRESPONDENCE
To the editor:
Granulocyte colony-stimulating factor receptor mutations in a
patient with acute lymphoblastic leukemia secondary to severe
congenital neutropenia
Severe congenital neutropenia (CN) is a group of hematopoietic
disorders with variable recessive inheritance characterized by absolute
neutropenia due to a maturation arrest of myeloid progenitor cells.
Patients with CN carry a predisposition toward the development of
myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML)
with an incidence of approximately 9%.1-2 Patients with
acquired nonsense mutations in the granulocyte colony-stimulating factor (G-CSF) receptor gene leading to the truncation of the membrane-distal region of the receptor have a high risk of leukemic transformation.2-3 So far, none of the known patients with
CN have developed a secondary acute lymphocytic leukemia (ALL). Here we
report on a 14-year-old girl with CN who developed a secondary pre-B
acute lymphoblastic leukemia (pre-B ALL). CN was diagnosed at the age
of 4 months, and she was started on r-metHuG-CSF (10 µg/kg/d) at age
9, followed by a prompt increase in neutrophil counts. Yearly bone
marrow investigations were consistent with CN (Figure
1A). At age 13, she developed an ALL with
more than 90% lymphoblasts in the bone marrow (Figure 1B). The
immunophenotype of the blasts as judged from flow cytometry was in
accordance with a pre-B ALL, coexpressing myeloid markers: CD19 (69%),
CD20 (57%), CD45 (68%), CD34 (84%), HLA-DR (91%), CD13 (67%), CD24 (22%), CD33 (29%), CD10 (< 1%). Intracellular staining
demonstrated TdT (67%), CD79a (20%), immunoglobulin M (µ chain)(20%), CD22 (10%), and MPO (< 2%). Immunostaining of bone
marrow sections confirmed these results and also revealed negative
staining for CD15, CD68, and lysozyme. Heteroduplex polymerase chain
reaction (PCR) analysis of genomic DNA to identify clonal
immunoglobulin and T-cell receptor gene rearrangements revealed an
incomplete immunoglobulin heavy-chain recombination reflecting a
DH-JH joining that did not involve a VH
segment (Figure 1C). Sequence analysis confirmed monoclonality of the
target and allowed identification of a DH3.22-JH6
rearrangement (Figure 1D). Since immature DH-JH recombinations are preferentially found in early precursor B-ALL malignancies, this result was consistent with the morphological and
immunological findings.
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| Figure 1.
Diagnosis of ALL secondary to CN.
Bone marrow smears at age 9 (A; severe congenital neutropenia before
start of G-CSF treatment) and at age 13 (B; severe congenital
neutropenia with secondary ALL). Heteroduplex PCR analysis (C) and
characterization of rearranged immunoglobulin and T-cell receptor gene
loci were performed as described earlier.5 Sequence
analysis revealed a monoclonal rearrangement of the DH-JH
type (D). Figures in brackets indicate the number of flanking
nucleotide deletions. Lowercase letters correspond to
N-nucleotides.
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By flow cytometry, we could demonstrate a G-CSF receptor
expression on the leukemic blasts (Figure
2). Sequencing of G-CSF receptor mRNA of
ALL lymphoblasts after subcloning of reverse transcriptase-polymerase
chain reaction (RT-PCR) products revealed point mutations similar to
those present in patients with CN/AML. In 8 of 15 clones (53%), we
detected a mutation at position 2414 of G-CSF receptor mRNA (E726X).
One clone (6%) demonstrated a stop mutation at position 2390 (E718X)
(Figure 2E). In 6 clones (40%) the wild-type sequence of the G-CSF
receptor mRNA was detected.
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| Figure 2.
Analysis of G-CSF receptor in lymphoblasts of the
patient.
FACS analysis of the lymphoblasts of the patient. The main
population of analyzed cells demonstrated a low-intensity staining for
CD19 (B). Minor populations corresponding to nonleukemic T or B cells
consisted of CD3+ cells (A) and CD19high cells
(B), respectively. Staining of the cells with anti-CD114 (anti-G-CSF
receptor) revealed a specific binding on about 80% of the cells (C).
Two-color analysis using FITC-labeled G-CSF and CD19-PE revealed a
specific binding of FITC-G-CSF on CD19low cells but not on
CD19high cells, indicating that the leukemic blast cells of
this patient are able to bind G-CSF (D). Schematic structure of the
cytoplasmic domain of the G-CSF receptor (G-CSFR) mRNA (E). The
nucleotide positions given below indicate the point mutations detected
in the patient reported here.
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We also examined peripheral blood neutrophils and mononuclear
cells at a complete remission status after chemotherapy treatment. In
both cell groups, the point mutation on nucleotide position 2414 was
detected in 2 of 10 clones (20%). None of the clones displayed a
mutation on nucleotide position 2390. Eighty percent (16 of 20) of the
analyzed clones showed wild-type sequence of the G-CSF receptor mRNA.
These data suggest that G-CSF receptor mutations were not restricted to
the malignant clone but were also present in a subpopulation of the
myeloid lineage. The mutation 2414 probably occured in a common
progenitor of the myeloid and B-cell lineage as one of the primary
transforming events in leukemogenesis in this patient. In contrast to
that, the mutation at position 2390 occurred later in a leukemic
subclone, maybe due to a general genetic instability of the G-CSF
receptor gene in CN. This would explain why the mutation 2414 is
detectable in both ALL-blasts and the neutrophils and mononuclear cells
at remission status of the patient.
To date, only myeloid leukemias secondary to CN have been
reported. To our knowledge this is the first case of ALL secondary to
CN. Interestingly, the G-CSF receptor mRNA of these leukemic cells
revealed a point mutation similar to mutations reported from patients
suffering from AML secondary to CN. This would support our hypothesis
that G-CSF receptor mutations are involved in
leukemogenesis.2-3 Indeed, 11 of 12 patients with MDS or
AML secondary to CN reveal G-CSF receptor point
mutations.2 The fact that there are patients with CN who
have not developed leukemia but express a mutated G-CSF receptor mRNA
strongly suggest that G-CSF receptor mutation is necessary but not
sufficient for the development of leukemia in CN
patients.2,4 Other genetic defects that seem to be important steps in leukemogenesis have already been reported in patients with CN/AML-like mutations in the ras gene or
monosomy 7.1,4 Indeed, the karyotype of our patient
demonstrated an unbalanced t(2;3) translocation and the deletion of 5q.
The contribution of these different events to leukemogenesis in CN
remains to be investigated.
Manuela Germeshausen, Matthias Ballmaier, Harald Schulze, and Karl Welte
Department of Pediatric Hematology and Oncology, Medical School
Hannover, Germany
Thomas Flohr
Institute of Human Genetics, University of Heidelberg, Germany
Klaus Beiske, Ingebjørg Storm-Mathisen, and Tore G. Abrahamsen
Departments of Pathology and Pediatrics, The National Hospital,
Oslo, Norway
Footnotes
Supported by grants from the Deutsche Krebshilfe
(10-1548-We2), Deutsche Forschungsgemeinschaft (We942/4-3), and
Norwegian Cancer Society.
References
1.
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].
2.
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.
3.
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;88:2369-2375.
4.
Kalra R, Dale D, Freedman M, et al.
Monosomy 7 and activating RAS mutations accompany malignant transformation in patients with congenital neutropenia.
Blood.
1995;86:4579-4586[Abstract/Free Full Text].
5.
Nakao M, Janssen JWG, Flohr T, Bartram CR.
Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by Lightcycler technology.
Cancer Res.
2000;60:3281-3289[Abstract/Free Full Text].
