Growth Factors

An acquired G-CSF receptor mutation results in increased proliferation of CMML cells from a patient with severe congenital neutropenia

Abstract

Severe congenital neutropenia (CN) is characterized by a maturation arrest of myelopoiesis at the promyelocyte stage. Treatment with pharmacological doses of recombinant human granulocyte colony-stimulating factor (rh-G-CSF) stimulates neutrophil production and decreases the risk of major infectious complications. However, approximately 15% of CN patients develop myeloid malignancies that have been associated with somatic mutations in the G-CSF receptor (G-CSFR) and RAS genes as well as with acquired monosomy 7. We report a CN patient with chronic myelomonocytic leukemia (CMML) who never received rh-G-CSF. Molecular analysis demonstrated a somatic G-CSFR mutation (C2390T), which led to expression of a truncated G-CSFR protein in the CMML. Normal G-CSFR expression was unexpectedly absent in primary and cultured CMML. In addition, CMML cells showed monosomy 7 and an oncogenic NRAS mutation. In vitro culture revealed a G-CSF-dependent proliferation of CMML cells, which subsequently differentiated along the monocytic/macrophage lineage. Our results provide direct evidence for the in vivo expression of a truncated G-CSFR in leukemic cells, which emerged in the absence of rh-G-CSF treatment and transduces proliferative signals.

Introduction

Severe congenital neutropenia (CN) is characterized by defective neutrophil maturation with an arrest of bone marrow myeloid progenitor cells at the promyelocyte and myelocyte stage of differentiation. Untreated patients suffer from recurrent life-threatening bacterial infections, deep tissue abscesses, and pneumonitis.1, 2 Recombinant human granulocyte colony-stimulating factor (rh-G-CSF) has revolutionized the care of individuals with CN; it acts by stimulating neutrophil production and thereby reduces the incidence and severity of infections.3, 4 Data from the International Registry for Severe Chronic Neutropenia (SCNIR) revealed that CN is a preleukemic disorder, with an estimated 15% risk of transformation to myelodysplastic syndrome (MDS) and acute leukemia.5 While most CN patients have developed MDS or acute myeloid leukemia (AML) in the context of prior rh-G-CSF treatment, few cases of AML were described in the pre-G-CSF era.6, 7 A potential interaction between rh-G-CSF treatment and leukemogenesis was suggested by the frequent finding of somatic point mutations within the intracellular region of the G-CSF receptor (G-CSFR) gene in CN patients with leukemia.8, 9, 10, 11 Most G-CSFR mutations introduce premature stop codons in place of glutamine residues between codons 715 and 732. Expressing these mutant alleles in myeloid cell lines enhances proliferation, inhibits maturation, confers resistance to apoptosis, and prolongs cell survival.12, 13 Increased G-CSF responsiveness and sustained receptor activation has been observed in mice carrying targeted G-CSFR mutations corresponding to those in CN patients.14, 15

From these in vitro and in vivo data and from the fact that somatic G-CSFR mutations are frequently detected prior to morphologic evidence of transformation, it has been hypothesized that they represent an important step in the progression of CN to MDS/leukemia.10, 11

Conversion to MDS/leukemia in CN patients has been associated with other cellular genetic abnormalities, for example partial or complete loss of chromosome 7 (7q− or monosomy 7), abnormalities of chromosome 21 (trisomy 21), and/or activating RAS mutations,5, 16 which are considered to act as cooperating events in the malignant transformation of leukemic progenitors. However, to date, there is no evidence that G-CSFR mutations contribute directly to malignant transformation.

We report a CN patient who developed chronic myelomonocytic leukemia (CMML) without antecedent rh-G-CSF treatment. Molecular analysis of mononuclear cells (MNCs) obtained at the diagnosis of CMML revealed somatic alterations including a mutation in the G-CSFR gene, expression of the truncated G-CSFR protein, no detectable expression of the normal G-CSFR, an oncogenic NRAS mutation, and monosomy 7. In vitro cultures of peripheral blood cells from this patient demonstrated a G-CSF-dependent proliferation of CMML cells expressing a truncated G-CSFR.

Case reports

Patient 1 developed recurrent bacterial infections in the first year of life. A bone marrow investigation at age 2 years revealed a growth arrest of myelopoiesis at the promyelocytic stage consistent with CN. Despite persistently low neutrophil counts, he never received rh-G-CSF. At age 18 years, he developed anemia and thrombocytopenia. CMML was diagnosed based on the finding of monocytosis greater than 1 × 109/l in peripheral blood, increased bone marrow monocytes, and dysplasia in all three cell lineages. A complete blood cell count showed the following values: hemoglobin 10.2 mg/dl (after transfusion), platelets 109 × 109/l, leukocytes 4.3 × 109/l with 5% segmented neutrophils, 1% basophils, 35% lymphocytes, 51% monocytes (2.2 × 109/l), 2% myelocytes and metamyelocytes, and 5% blasts. A bone marrow biopsy showed dysplastic granulopoiesis and erythropoiesis, minor dysplasia in megakaryopoiesis and monocytosis and less than 30% blasts. Cytogenetic analysis disclosed monosomy 7 in 22 out of 25 unstimulated metaphase cells. The patient also had leukemic skin infiltrates, with a high proportion of cells positive for macrophage markers and a high proliferative capacity. He received two courses of chemotherapy, followed by bone marrow transplantation from an unrelated HLA-identical donor. He has been in remission with normal peripheral blood cell counts for the past 4 years.

As a control, three other patients (patients 2–4) with sporadic CMML were analyzed for G-CSF receptor mutations.

Materials and methods

Purification of mononuclear cells and neutrophil granulocytes

MNCs and granulocytes have been isolated from fresh heparinized blood. Blood was mixed with hydroxy ethyl starch (Plasmasteril, Fresenius AG) and allowed to sediment for 30 min at room temperature. The leukocyte-rich supernatant was layered on Ficoll–Isopaque. After centrifugation, MNCs were recovered from the interface and neutrophil granulocytes were obtained as sedimented cell fraction and further depleted of contaminating erythrocytes by hypotonic lysis.

Molecular analyses of the G-CSFR gene and G-CSFR mRNA

RNA and DNA were extracted from the neutrophils and MNCs of peripheral blood from patients 1–4 and from cultured MNCs of patient 1. Total RNA was isolated using the guanidium thiocyanate method17 and treated with DNAse. RNA (1 μg) was primed with oligo (dT) and reverse described into cDNA in a 20 μl reaction volume containing 200 U reverse transcriptase (MMLV). Genomic DNA was prepared by using QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's guidelines. A 256 bp fragment including the mutation-sensitive region 2384–2429 was amplified by PCR from genomic DNA or from cDNA after reverse transcription of mRNA as described previously.9 One-tenth of RT reaction mixture or 100–200 ng of genomic DNA was used for PCR amplification in a 25 μl volume containing 0.2 μ M of each primer, 0.16 μ M of dNTPs, 1 U TAQ/Pfu polymerase mixture, and 1 × standard Taq buffer (Roche). PCR primers were designed according to the published sequence of the G-CSFR cDNA18 (acc. no. M59818) and were as follows: FW IndexTerm5′-aac agc tca gag acc tgt ggc ctc (nucleotides 2306–2329), RV IndexTerm5′-cca agg ggc tgg cct gga acc aga (nucleotides 2538–2561). PCR amplification was performed for 45 cycles consisting of 30 s at 95°C, 30 s at 66°C, and 30 s at 72°C. After amplification, PCR products were separated on agarose gels, purified with StrataPrep (Stratagene), and ligated to the SrfI site of pBluescript vector (Stratagene). The PCR products were sequenced after subcloning. This procedure we have repeated seven times for the investigation of three independently prepared RNAs and two times each for the genomic DNA and the DNA isolated from cells after culture for 14 days. In total, we analyzed 28 clones generated from neutrophil mRNA and 52 clones generated from peripheral blood MNC (PB-MNC) mRNA. In addition, we analyzed 42 clones generated from the genomic DNA from PB-MNCs and 51 clones generated from genomic DNA of cells cultured for 14 days with G-CSF.

Analysis of G-CSFR protein expression

PB-MNCs of patient 1 at the time point of diagnosis of CMML were lysed and immunoprecipitated with a monoclonal anti-G-CSF receptor antibody (CD114, clone 129).19 G-CSF receptor immunoblot analysis was performed with the same antibody. LGM-1 cells (murine myeloid cell line) that were stably transfected with different G-CSF receptor expression vectors leading to surface expression of distinct truncated G-CSF receptor proteins served as controls.20

In vitro cultures of peripheral blood cells

PB-MNCs of patient 1 at the time point of diagnosis of CMML were cultivated in Iscove's modified Dulbecco's medium (IMDM) with 30% fetal bovine serum and with or without 50 ng/ml rh-G-CSF (Amgen). Cultures were analyzed on days 7 and 14 for morphology, surface marker expression, monosomy 7, and G-CSFR mutations. Cultures of PB-MNCs of normal donors served as controls for morphologic and surface marker expression analyses.

Flow cytometric analyses and cell sorting

MNCs from patient 1 and cultured cells from the patient and from healthy controls at days 7 and 14 were analyzed for light scatter properties and for surface expression of the monocytic marker CD14 (clone TüK4, Caltag) and of the G-CSFR (CD114, clone LMM741, Pharmingen) using a fluorescence-activated cell sorter (MoFlo, Cytomation). Viability was assessed by exclusion of propidium iodide (PI). On days 7 and 14 of cell culture, cells of interest were sorted and analyzed as described.

Analysis of RAS genes

Single-strand conformational polymorphism (SSCP) analysis of exons 1 and 2 of the NRAS and KRAS2 genes was performed from PB-MNCs of patient 1 at the time point of diagnosis of CMML and from a bone marrow smear of this patient at age 2 years as previously described.16

Chromosome 7 FISH

FISH analysis was performed on cytospin preparations according to the procedure published earlier.21

Results and discussion

Primary CMML cells of patient 1 contain an acquired G-CSFR mutation and wild-type mRNA was not detectable

Sequence analysis of cDNA prepared from patient's neutrophils and MNCs obtained at the diagnosis of CMML revealed a nonsense G-CSFR mutation (C2390T) that resulted in loss of 95 carboxy-terminal amino acids of the receptor (Figure 1a). The mutation was confirmed by sequencing genomic DNA extracted from the same MNCs. Analysis of DNA samples from the parents and from a bone marrow specimen obtained when the patient was 2 years old revealed the normal sequence, proving that the mutation was acquired and somatic. Three patients diagnosed with de novo CMML showed the normal G-CSFR sequence, which confirms previous findings that G-CSFR mutations are correlated with malignant transformation of CN but not with primary myeloid malignancies. Surprisingly, and in contrast to other reports of G-CSFR mutations in CN, no normal G-CSFR mRNA was detectable in the patient's MNCs (0/52 clones) and neutrophils (0/28 clones). However, 12 of 42 clones amplified from DNA extracted from these MNCs contained the normal G-CSFR sequence, whereas 30 showed the mutant sequence.

Figure 1
figure1

G-CSF receptor mutation and expression of a truncated G-CSF receptor in patient 1. (a) The cartoon depicts the results of G-CSFR mRNA analysis and the postulated effect on the G-CSFR protein. The analyzed PCR product with the adjacent primers is depicted above the 3′-terminus of G-CSFR mRNA derived from exons 14–17. The C2390T mutation leads to an exchange of glutamine into a stop codon and a truncated protein of 718 amino acids (aa), compared to 813 aa in the normal protein. The gray boxes in the intracytoplasmatic domain represent boxes 1–3. Examples of PCR products used for cloning and sequence analysis are shown in (b–d). (b) PCR product from PB-MNCs derived DNA from patient 1 at time point CMML. (c) cDNA generated from PB-MNC RNA at time point CMML from patient 1 and controls. (d) DNA from PB-MNCs cultured for 14 days with 50 ng/ml G-CSF (see text). −: negative control; M: molecular weight marker. The marker shown in (b–d) represent fragments of 653, 517, 453, 394, 298, and 234 bp. (e–g) Protein analysis: comparison of wild-type receptor (813 aa) with a construct encoding a truncated protein (737 aa) and the truncated G-CSFR of patient 1 as postulated from the molecular analysis. (e) Lysates from neutrophil granulocytes (wt) and from LGM-1 cells stably transfected with constructs leading to G-CSFR molecules of the indicated length were immunoprecipitated and detected with a monoclonal anti-G-CSFR antibody. (f) Comparison of G-CSFR from MNCs of patient 1 with the construct S737 (immunoprecipitation and Western blot detection as in (b). (g) Flow cytometric detection of the surface expression of the G-CSFR on CMML cells. dotted line: isotype control; solid line: anti-CD114.

From these results, we could not decide whether the detection of exclusive expression of mutated G-CSFR mRNA was due to a preferential expression of a heterozygously mutated allele or due to a loss of the normal G-CSFR allele in the leukemic clone.

Acquired mutations in the G-CSFR gene have been reported in a subset of patients with CN progressing to acute leukemia or MDS, suggesting a contribution of these mutations to leukemogenesis.9, 12 An important aspect of the current case is that our patient acquired a G-CSFR mutation that achieved clonal dominance in the absence of any treatment with rh-G-CSF. The G-CSFR mutations detected in patients are predicted to lead to the expression of a truncated receptor lacking the carboxy-terminus of the intracellular domain that is critical for maturation and growth arrest signaling. The C2390T mutation is not specifically associated with CMML, since it has been found in other CN patients who evolved to acute myeloid and acute lymphoid leukemia.10, 22 In contrast to previously described cases, we found a virtually exclusive expression of the mutated allele. To date, no data exist on the importance of the balance between normal and mutant G-CSFR mRNA and receptor proteins, respectively. Studies using the murine myeloid 32D cell line engineered to coexpress normal and mutant G-CSFR proteins suggest a dominant effect of the mutant protein.12 Coexpression of normal and mutant G-CSFR forms in myeloid cell lines interfered with terminal maturation induced by the normal receptor and led to hyperproliferative responses to G-CSF.8, 12, 23 Mice that are heterozygous for a mutant ‘knock-in’ allele of the murine G-CSFR are also hyper-responsive to rh-G-CSF in vivo.14 However, these results do not preclude the possibility that a subclone that has acquired a G-CSFR mutation might gain an additional proliferative advantage by inactivating the normal allele. For example, cell lines that express oncogenic RAS tend to amplify the mutant allele,24 and recent data in a murine lung cancer model characterized by alkylator-induced KRAS2 point mutations have shown that absence of the wild-type KRAS2 allele dramatically accelerates the disease phenotype.25 Recently, a patient with juvenile myelomonocytic leukemia was reported with both an oncogenic mutation in the PTPN11 gene and loss of the normal allele.26 Monoallelic expression of a de novo mutated gene has been described earlier,27 and seems to be a common feature for the expression of the tumor suppressor gene p73 in de novo AML.28 Our data are compatible with either somatic deletion of the normal G-CSFR allele in a subset of circulating CMML MNCs or with epigenetic inactivation of the normal allele.

A truncated G-CSF receptor is expressed on monocytic cells of the CMML

We next asked whether the mutated G-CSFR mRNA is translated in vivo. So far, no truncated G-CSFR protein in patients with heterozygous G-CSFR mRNA expression could be detected by immunoblotting, suggesting that the mRNA might be unstable.29 As we detected no wild-type G-CSFR mRNA in the PB-MNCs of patient 1, we expected to detect solely the truncated protein. According to the sequence analysis, this protein would even be smaller than the previously described S737 mutant.20 First, we confirmed that wild-type G-CSFR protein migrates slower on an SDS electrophoresis than the terminally truncated proteins S737, S761, and S783 (Figure 1e). We next compared the molecular weight of the S737 mutant with the G-CSFR precipitated from MNCs obtained from patient 1 at the diagnosis of CMML. As expected, we detected a faster migrating protein in accordance with the predicted molecular weight (718 amino acids). No additional immunoreactive band was detectable above the 116 kDa marker band (Figure 1f). Taken together with the results of molecular analyses, we can therefore conclude that a mutated G-CSFR mRNA is indeed translated into a truncated G-CSFR protein in primary cells. The G-CSFR protein was also detectable by flow cytometric analysis on the surface of the monocytic cells including the leukemic CMML cells, excluding an internal accumulation of the mutated protein (Figure 1g). This is the first report demonstrating that a truncated G-CSFR protein is in fact expressed in vivo. Our data further suggest that the normal G-CSFR protein is absent from this patient's CMML cells.

In vitro response to recombinant G-CSF

We next asked whether this mutant G-CSF receptor was functional and could modulate cellular responses. The patient's PB-MNCs were cultured, both with and without rh-G-CSF as the only growth factor. In the cultures containing rh-G-CSF, we observed small clusters of relatively large, dividing cells (Figure 2g). Flow cytometric analysis on day 7 revealed a population of cells homogenous in scatter properties (medium forward scatter and low side scatter) but consisting of CD14+ and CD14 cells (Figure 2f and h). For further characterization of these cells, we sorted CD14 high expressing cells (sorting regions A and C; Figure 2f and h) and CD14-negative cells (sorting regions A and B). We confirmed monocytic morphology by Giemsa staining on cytocentrifuged cells and performed karyotype analysis using FISH. CD14 high expressing cells had a monocytic morphology, and the CD14 cells were immature myeloblasts (Figure 2e, insets). Both populations demonstrated monosomy 7 in all cells analyzed, arguing for a common progenitor and continuous maturation of immature cells of the leukemic clone to monocytic CMML cells. During the second week in culture, the percentage of CD14+ cells increased to over 80%. The cells differentiated into large cells of monocyte/macrophage morphology, which constitute a nearly uniform population at day 14 (Figure 2i–l). These cells (100%) revealed monosomy 7 by FISH analysis (Figure 2n) and expressed the G-CSFR on the cell surface as proven by flow cytometry (Figure 2m). In contrast to the results on day 0, wild-type G-CSFR was not detectable in genomic DNA extracted from the sorted CD14+ population on day 14 (0 out of 51 clones), arguing for a loss of the wild-type allele in the leukemic progenitors.

Figure 2
figure2

In vitro culture of MNCs of patient 1 with or without rh-G-CSF. MNCs of the patient were cultured with or without G-CSF as indicated in the text and analyzed on day 7 (a–d: no G-CSF; e–h: 50 ng/ml G-CSF) and day 14 (i–n: 50 ng/ml G-CSF). Cytospins from the indicated cultures were stained with May–Gruenwald–Giemsa (a, e, i) and were analyzed flow cytometrically for their light scatter properties (b, f, j), for viability (PI exclusion), and for CD14 expression (d, h, l: CD14 staining of PI-negative cells). Phase contrast light microscopy of the cultures (c, g, k) demonstrated small clusters of proliferating cells on day 7 (g). CD14+/PI (h, region C) and CD14/PI cells (h, region B) of the cell population with medium forward scatter and low side scatter (f, region A) were sorted on day 7 for further analysis (see text). CD14-positive cells from day 14 expressed G-CSFR as demonstrated by double staining with CD114 (m) and showed one chromosome 7 signal per cell as demonstrated by FISH (n).

In contrast, in cultures lacking G-CSF, most cells died during the first 7 days of culture (Figure 2a–d). The viability was about 20% compared to the presence of G-CSF and there was no expansion of immature cells on day 7 (Figure 2a). The culture consisted mainly of lymphocytes and mature monocytes/macrophages as judged by morphology; karyotype analysis revealed disomy 7 in all living cells analyzed. The cells also differentiated along the monocytic/macrophage lineage, suggesting that this process is growth factor-independent and addition of rh-G-CSF resulted in an expansion rather than in differentiation of progenitor cells.

As a control, we cultivated PB-MNCs from normal donors (n=4), both with and without G-CSF. The cell number dropped markedly over the first 7 days of culture independent of the addition of rh-G-CSF (data not shown), and the percentage of CD14+ cells decreased to below 10% of the value at day 0. In the presence of G-CSF, a slightly higher proportion of cells became CD14 positive compared to cultures lacking G-CSF. However, there was no proliferation of either CD14+ or CD14 cells in both cultures.

From these results, we conclude that immature leukemic progenitors expressing a truncated G-CSFR protein expanded in response to G-CSF. The immature leukemic CD14 cells further differentiated along the monocytic line (CD14+), as shown by detection of monosomy 7 and the expression of the truncated receptor in all cell populations analyzed on day 14. The following reasons could promote development of the blast cells along the monocytic lineage: (1) a maturation arrest at the stage of promyelocytes as a result of the underlying disease CN, which prevents further differentiation along the neutrophil lineage, (2) a lack of differentiation signals from the truncated receptor, (3) a commitment of the leukemic progenitors to the monocytic lineage, or (4) coexistence of a RAS mutation (see below), which is associated with monocytic differentiation in mouse models of CMML.30, 31 Interestingly, monocytosis is a common feature of CN, independent of rh-G-CSF treatment.1

Our observation that myeloid progenitor cells expressing a truncated G-CSFR are able to proliferate in response to G-CSF principally confirms the conclusions from previous experiments using transfected cell lines or transgenic mice. It supports the hypothesis that a truncated receptor induces a hyperproliferative response and that G-CSF can provide a selective pressure to expand cell clones bearing G-CSFR mutations. However, unlike 32D or Ba/F3 cells transduced with mutant G-CSFR constructs,12, 13 our patient's cells were unable to survive in the absence of growth factors. The absence of previous treatment with rh-G-CSF argues that exogenous rh-G-CSF did not contribute to acquisition of G-CSFR mutations. Levels of endogenous G-CSF are elevated in CN32 and seem to be sufficiently high to promote this process.

An oncogenic NRAS mutation in CMML cells might act cooperatively to the G-CSFR mutation

Tumorigenesis is understood as a process in which multiple steps are involved in the transition to a leukemic cell.33 Activating RAS mutations are – besides monosomy 7 – associated with malignant transformation in CN.1, 5, 16 Therefore, we screened for mutations in exons 1 and 2 of KRAS2 and NRAS genes in the CMML patient's specimens.

SSCP analysis of DNA extracted from blood leukocytes revealed abnormal migration of a fragment amplified from NRAS exon 1 that was not present in the bone marrow sample obtained at age 2 (Figure 3a). The products were cloned and sequence analyses confirmed a GGT (glycine) to CGT (arginine) substitution at codon 13 (Figure 3b), which is one of the known activating mutations of NRAS.34 No anomalies were found in the SSCP analysis of KRAS2.

Figure 3
figure3

Analysis of NRAS gene. (a) SSCP analysis of DNA for abnormalities in exon 1 of NRAS. 1: MNCs of patient 1 at time point CMML, 2: patient's bone marrow at age 2 years, 3: healthy control. (b) Sequence analysis of cloned abnormal PCR products showed a G-to-C transition in the first position of codon 13 of NRAS.

The Ras–mitogen-activated protein (MAP) kinase pathway is a downstream effector of the activated G-CSFR, which is predominantly activated by membrane-proximal domains.35, 36 Since Ras is an effector of the activated G-CSFR, truncated receptors might contribute to leukemogenesis, at least in part, through effects on Ras. However, although mutant G-CSFR proteins induce prolonged activation of STAT pathways and showed defective internalization in cell lines, Ras signaling appears normal.15, 37 The presence of both G-CSFR and NRAS mutations in the current patient argues that these lesions are nonredundant and cooperate in leukemogenesis in agreement with the ‘multiple hit’ theory. Studies of human malignancies have identified NRAS codons 12, 13, and 61 as the most frequent sites for oncogenic mutations, resulting in inhibition of intrinsic GTPase activity and inappropriate high-level activation of downstream effectors that mediate proliferation and viability. Activating mutations of NRAS lead to the growth factor-independent proliferation of hematopoietic cells and can also result in their accumulation due to reduced levels of apoptosis; both mechanisms may potentially lead to the leukemic phenotype.38, 39 Since the G-CSFR mutation was not exclusively present in the CMML cells but was present in at least a vast majority of mature neutrophils in the peripheral blood, we have to assume that this mutation has been acquired by an early myeloid precursor cell providing it with a proliferative advantage and leading to a clonal expansion of cells bearing the G-CSFR mutation. Importantly, since RAS mutations have only been found in CN patients after evolution to MDS or AML, it is likely that they represent a rather late event in leukemogenesis. Based on the high incidence of RAS mutations in patients with CMML and on recent data from mouse models showing that somatic activation of an oncogenic allele of Kras2 induces a fatal myeloproliferative disease,30, 31 we speculate that the NRAS mutation in this patient might have contributed to the CMML phenotype.

Conclusions

In summary, we describe a patient with CN who was not treated with rh-G-CSF, yet developed CMML associated with a truncating mutation in the G-CSFR allele and loss of the wild-type allele. The mutant receptor was expressed on his CMML cells, which proliferated and survived in response to rh-G-CSF. The finding of an activating NRAS mutation and monosomy 7 in the leukemic clone provides direct evidence that these lesions cooperate with somatic G-CSFR mutations in leukemogenesis.

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Acknowledgements

We thank Sabine Jakobs and Klara Cherkaoui for excellent technical assistance and Gernot Beutel for his help with the microscopic pictures. The work was supported, in part, by Deutsche Krebshilfe (10-1548-We2) and by NIH Grant R01 CA72614.

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Correspondence to M Germeshausen.

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Germeshausen, M., Schulze, H., Kratz, C. et al. An acquired G-CSF receptor mutation results in increased proliferation of CMML cells from a patient with severe congenital neutropenia. Leukemia 19, 611–617 (2005). https://doi.org/10.1038/sj.leu.2403663

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Keywords

  • G-CSF receptor
  • RAS mutations
  • congenital neutropenia
  • CMML
  • leukemogenesis

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