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.
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.
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.
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.
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.
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.
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.
Welte K, Boxer LA . Severe chronic neutropenia: pathophysiology and therapy. Semin Hematol 1997; 34: 267–278.
Zeidler C, Boxer L, Dale DC, Freedman MH, Kinsey S, Welte K . Management of Kostmann syndrome in the G-CSF era. Br J Haematol 2000; 109: 490–495.
Bonilla MA, Gillio AP, Ruggeiro M, Kernan NA, Brochstein JA, Abboud M et al. Effects of recombinant human granulocyte colony-stimulating factor on neutropenia in patients with congenital agranulocytosis. N Engl J Med 1989; 320: 1574–1580.
Welte K, Gabrilove J, Bronchud MH, Platzer E, Morstyn G . Filgrastim (r-metHuG-CSF): the first 10 years. Blood 1996; 88: 1907–1929.
Freedman M, Bonilla MA, Fier C, Bolyard AA, Scarlata D, Boxer LA et al. Myelodysplasia syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy. Blood 2000; 96: 429–436.
Gilman PA, Jackson DP, Guild HG . Congenital agranulocytosis: prolonged survival and terminal acute leukemia. Blood 1970; 36: 576–585.
Rosen RB, Kang SJ . Congenital agranulocytosis terminating in acute myelomonocytic leukemia. J Pediatr 1979; 94: 406–408.
Dong F, Hoefsloot LH, Schelen AM, Broeders LCAM, Meijer Y, Veerman AJP et al. Identification of a nonsense mutation in the granulocyte-colony-stimulating factor receptor in severe congenital neutropenia. Proc Natl Acad Sci USA 1994; 91: 4480–4484.
Tidow N, Pilz C, Teichmann B, Müller-Brechlin A, Germeshausen M, Kasper 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.
Germeshausen M, Ballmaier M, Welte K . Implications of mutations in hematopoietic growth factor receptor genes in congenital cytopenias. Ann NY Acad Sci 2001; 938: 305–320; discussion 20, 21.
Tschan CA, Pilz C, Zeidler C, Welte K, Germeshausen M . Time course of increasing numbers of mutations in the granulocyte colony-stimulating factor receptor gene in a patient with congenital neutropenia who developed leukemia. Blood 2001; 97: 1882–1884.
Dong F, Russell KB, Tidow N, Welte K, Löwenberg 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.
Hunter MG, Avalos BR . Granulocyte colony-stimulating factor receptor mutations in severe congenital neutropenia transforming to acute myelogenous leukemia confer resistance to apoptosis and enhance cell survival. Blood 2000; 95: 2132–2137.
McLemore ML, Poursine-Laurent J, Link DC . Increased granulocyte colony-stimulating factor responsiveness but normal resting granulopoiesis in mice carrying a targeted granulocyte colony-stimulating factor receptor mutation derived from a patient with severe congenital neutropenia. J Clin Invest 1998; 102: 483–492.
Hermans MHA, Antonissen C, Ward AC, Mayen AEM, Ploemacher RE, Touw IP . Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene. J Exp Med 1999; 189: 683–692.
Kalra R, Dale D, Freedman M, Bonilla MA, Weinblatt M, Ganser A et al. Monosomy 7 and activating RAS mutations accompany malignant transformation in patients with congenital neutropenia. Blood 1995; 86: 4579–4586.
Chomczynski P, Sacchi N . Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 1987; 162: 156–159.
Fukunaga R, Seto Y, Mizushima S, Nagata S . Three different mRNAs encoding human granulocyte colony-stimulating factor receptor. Proc Natl Acad Sci USA 1990; 87: 8702–8706.
Kasper B, Welte K, Hadam MR . MC24 CD114 (granulocyte-colony stimulating factor receptor) Workshop Panel report. In: Kishimoto T, Kikutani H, von dem Borne AEGKr, Goyert SM, Mason DY, Myasaka M, Moretta L, Okumura K, Shaw S, Springer TA, Sugamura K, Zola H (eds). Leucocyte Typing VI White Cell Differentiation Antigens. New York & London: Garland Publishing Inc., 1997, pp 1072–1074.
Herbst A, Koester M, Wirth D, Hauser H, Welte K . G-CSF receptor mutations in patients with severe congenital neutropenia do not abrogate Jak2 activation and stat1/stat3 translocation. Ann NY Acad Sci 1999; 872: 320–325; discussion 5–7.
Wilkens L, Flemming P, Gebel M, Bleck J, Terkamp C, Wingen L et al. Induction of aneuploidy by increasing chromosomal instability during dedifferentiation of hepatocellular carcinoma. Proc Natl Acad Sci USA 2004; 101: 1309–1314.
Germeshausen M, Ballmaier M, Schulze H, Welte K, Flohr T, Beiske K et al. Granulocyte colony-stimulating factor receptor mutations in a patient with acute lymphoblastic leukemia secondary to severe congenital neutropenia. Blood 2001; 97: 829–830.
Dong F, Buitenen Cv, Pouwels K, Hoefsloot LH, Löwenberg B, Touw IP . Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation. Mol Cell Biol 1993; 13: 7774–7781.
Finney R, Bishop J . Predisposition to neoplastic transformation caused by gene replacement of H-ras1. Science 1993; 260: 1524–1527.
Zhang Z, Wang Y, Vikis HG, Johnson L, Liu G, Li J et al. Wildtype Kras2 can inhibit lung carcinogenesis in mice. Nat Genet 2001; 29: 25–33.
Loh ML, Vattikuti S, Schubbert S, Reynolds MG, Carlson E, Lieuw KH et al. Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 2004; 103: 2325–2331.
Miraglia Del Giudice E, Lombardi C, Francese M, Nobili B, Conte ML, Amendola G et al. Frequent de novo monoallelic expression of β-spectrin gene (SPTB) in children with hereditary spherocytosis and isolated spectrin deficiency. Br J Haematol 1998; 101: 251–254.
Stirewalt D, Clurman B, Appelbaum F, Willman C, Radich J . p73 mutations and expression in adult de novo acute myelogenous leukemia. Leukemia 1999; 13: 985–990.
Kasper B, Herbst A, Pilz C, Germeshausen M, Tidow N, Hadam MR et al. Severe congenital neutropenia patients with point mutations in the granulocyte colony-stimulating factor (G-CSF) receptor mRNA express a normal G-CSF receptor protein. Blood 1997; 90: 2839–2840.
Braun BS, Tuveson DA, Kong N, Le DT, Kogan SC, Rozmus J et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci USA 2004; 101: 597–602.
Chan IT, Kutok JL, Williams IR, Cohen S, Kelly L, Shigematsu H et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest 2004; 113: 528–538.
Mempel K, Pietsch T, Menzel T, Zeidler C, Welte K . Increased serum levels of granulocyte colony-stimulating factor in patients with severe congenital neutropenia. Blood 1991; 77: 1919–1922.
Kelly LM, Gilliland DG . Genetics of myeloid leukemias. Annu Rev Genomics Hum Genet 2002; 3: 179–198.
Hirai H, Kobayashi Y, Mano H, Hagiwara K, Maru Y, Omine M et al. A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome. Nature 1987; 327: 430–432.
Nicholson SE, Novak U, Ziegler SF, Layton JE . Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44 MAPK. Blood 1995; 86: 3698–3704.
Dong F, Larner AC . Activation of Akt kinase by granulocyte colony-stimulating factor (G-CSF): evidence for the role of a tyrosine kinase activity distinct from janus kinases. Blood 2000; 95: 1656–1662.
Barge RMY, Koning JP, Pouwels K, Dong F, Löwenberg B, Touw IP . Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G-CSF-mediated activation of signaling complexes of the p21ras route. Blood 1996; 87: 2148–2153.
Bartram CR . Mutations in ras genes in myelocytic leukemias and myelodysplastic syndromes. Blood Cells 1988; 14: 533–538.
Shih L-Y, Huang C-F, Wang P-N, Wu J-H, Lin T-L, Dunn P et al. Acquisition of FLT3 or N-ras mutations is frequently associated with progression of myelodysplastic syndrome to acute myeloid leukemia. Leukemia 2004; 18: 466–475.
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.
About this article
Cite this article
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
- G-CSF receptor
- RAS mutations
- congenital neutropenia
Clinical Immunology (2018)
Nature Reviews Disease Primers (2017)
Prevalence and impact of colony stimulating factor 3 receptor (CSF3R) mutations among Egyptian acute myeloid leukemia patients
Leukemia Research (2014)
Frontiers in Oncology (2014)