Frequency of Point Mutations in the Gene for the G-CSF Receptor in Patients with Chronic Neutropenia Undergoing G-CSF Therapy

Transcription

1 Frequency of Point Mutations in the Gene for the G-CSF Receptor in Patients with Chronic Neutropenia Undergoing G-CSF Therapy NICOLA TIDOW, CHRISTINA PILZ, BRIGITTE KASPER, KARL WELTE Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany Key Words. Severe congenital neutropenia G-CSF receptor mutations. G-CSF treatment ABSTRACT Point mutations in the gene for the G-CSF receptor have been reported previously in a subgroup of patients with severe congenital neutropenia. Here, we investigated the frequency of these specific G-CSF receptor mutations in patients with neutropenic disorders undergoing treatment with recombinant human (r-methu)g-csf (Filgrastim). Nucleotides 236 to 2561, including the critical region (nucleotides ) from the intracellular domain of the G-CSF receptor gene, were amplified by reverse transcriptase-polymerase chain reaction, and DNA was sequenced directly and after transformation in E. coli. Four of 3 patients with severe congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. Two of the four patients with a mutated G-CSF receptor developed acute myeloid leukemia secondary to congenital neutropenia. G-CSF receptor analyses were performed in myeloid cells taken at different time points in the four patients with the mutated receptor, and no correlation between occurrence of the mutation and time or dose of r-methug-csf treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 26 congenital neutropenia patients. Additionally, no G-CSF receptor point mutations could be seen in neutrophils, blood and bone marrow mononuclear cells from patients with cyclic or idiopathic neutropenia, and bone marrow mononuclear cells from patients suffering from severe aplastic anemia. Similar results were obtained by Touw et al., demonstrating that five out of 25 patients with congenital neutropenia reveal G-CSF receptor mutations. These data show that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur only in a subgroup of severe congenital neutropenia patients. Furthermore, our data suggest that the described G-CSF receptor point mutations are not correlated with the start, duration or doses of r-methug-csf treatment, but might result from genetic instability in the G-CSF receptor gene in severe congenital neutropenia. Stem Cells 1997;15(suppl 1): INTRODUCTION Chronic neutropenia represents a heterogenous group of neutropenic diseases including severe congenital, cyclic and idiopathic neutropenia. Severe congenital neutropenia, or Kostmann s syndrome, is characterized by a maturation arrest of neutrophil precursors at the level of promyelocytes or myelocytes in the bone marrow [I, 21. The ANC are below 2/p1 in peripheral blood. Congenital neutropenia Hematopoietic Stem Cells. STEM CELLS 1997;15(suppl 1): AlphaMed Press. All rights reserved.

2 114 G-CSF Receptor Mutations predisposes a patient to frequent and severe bacterial infections. Several clinical studies demonstrated that treatment of congenital neutropenia patients with recombinant human G-CSF (r-methug-csf, Filgrastim, 1-12 pg/kg/d) results in an increase in circulating neutrophils and a reduction in infectionrelated events in more than 95% of the patients [3-61. Patients with cyclic neutropenia show 21-day oscillations in their ANC with at least four days of severe neutropenia (ANC below 2/p1). Again treatment with r-methug-csf (3 pg/kg/d) leads to an increase in ANC and a decrease in the number of bacterial infections. Idiopathic neutropenia might be potentially caused by viral infections, toxins or drugs. The degree to which patients suffered from bacterial infections correlated to the degree of neutropenia. The International Registry for Severe Chronic Neutropenia has collected data on 249 patients with severe congenital neutropenia, 97 patients with cyclic neutropenia and 16 patients with idiopathic neutropenia [7, 81. Between 1988 and 1996, 23 of the 249 congenital neutropenia patients developed myelodysplastic syndrome or acute myeloid leukemia (MDS/AML). None of the registered patients with cyclic or idiopathic neutropenia developed MDS/AML, suggesting that r-methug-csf is not involved in leukemogenesis, and severe congenital neutropenia might represent a premalignant disorder of myelopoiesis [7-91. Severe aplastic anemia represents another example of a preleukemic disease. It is characterized by pancytopenia including severe neutropenia. Most patients can be effectively treated by bone marrow transplantation or immunosuppressive therapy. In most severely neutropenic aplastic anemia patients, combined therapy with r-methug-csf results in improvement in ANC and protection against infection. However, the risk of developing MDS or AML is significantly higher in these patients. The pathophysiology of the different forms of chronic neutropenia is still unknown. Defects in G-CSF-mediated signal transduction have been proposed, and increased tyrosine phosphorylation and activation of the receptor-associated protein tyrosine kinase JAK2 could be shown in neutrophils from patients with congenital neutropenia [lo]. Recently, point mutations at nucleotide positions 239 and 2429 in the intracellular region of the G-CSF receptor gene have been described and linked to the development of secondary AML in five patients with congenital neutropenia [ll, 121. Herein, we tested patients with chronic neutropenia and patients with severe aplastic anemia for the occurrence of point mutations in the critical region of the cytoplasmic domain of the G-CSF receptor (nucleotides ) in order to answer the following questions: A) what is the frequency of G-CSF receptor mutations in chronic neutropenia; B) are point mutations in the intracellular part of the G-CSF receptor detectable in other forms of preleukemic disorders, for example severe aplastic anemia, and C) what role does G-CSF play in the development of MDS or AML? MATERIALS AND METHODS Separation of Myeloid Cells Neutrophils and mononuclear cells (mainly lymphocytes and monocytes/macrophages) have been is@ lated from fresh heparinized blood of healthy volunteers or neutropenic patients by dextran sedimentation, Ficoll density centrifugation and hypotonic lysis of residual erythrocytes. More than 98% of the cells were viable as assayed by trypan blue dye exclusion. In addition, in some patients mononuclear cells from the bone marrow were used for the G-CSF receptor analyses. Isolation of DNA and RNA Genomic DNA was isolated from neutrophils and mononuclear cells using spin columns and buffers from the QIAamp tissue kit (Qiagen; Chatsworth, CA). Total RNAs were extracted from the cells according to the single-step isolation method [13] using RNAzol B (WAK-Chemie Medical GmbH; Bad Homburg, Germany).

3 Tidow, Pilz, Kasper et al. 115 Polymerase Chain Reaction (PCR) [14,15] RNA has been transcribed into cdna with reverse transcriptase (1 units/pg RNA, AMV reverse transcriptase, Promega; Madison, WI) using random primers (.5 pg/pg RNA) in 5 mm Tris-HCI, 5 mm KCl, 1 mm MgC12, 1 mm DTT,.5 mm spermidine and 1 mm dntps at 37 C for 15 min and at 42 C for 45 min. PCR was performed with specific primers amplifying nucleotides or of the cytoplasmic domain of the G-CSF receptor. The numbering of the nucleotides refers to Fukunaga and colleagues [16]. The critical region of the G-CSF receptor is defined from nucleotide and includes all mutations that have been reported thus far. Amplification of cdna or DNA templates was carried out in 25 p1 of 1 mm Tris-HC1,5 mm KC1, 1 mm MgC12,.1 pm primer and 1 unit of Taq Polymerase (Boehringer Mannheim; Germany) for 25 cycles in a thermocycler (VariusV; Landgraf, Langenhagen, Germany). Each cycle consisted of denaturation for 6 sec at 94"C, annealing for 1 sec at 64 C and extension for 1 sec at 72 C. Fragments were analyzed on agarose gels stained with ethidium bromide. Nonradioactive DNA Sequencing Individual bands of PCR products were either sequenced directly or after purification via low melting gel electrophoresis, ligation into a PCR-script SK(+) plasmid (Stratagene; La Jolla, CA) and cloning in E. coli by the dideoxynucleotide chain-termination method [ 171. Cycle sequencing was performed with DIG-labeled primer using the DIG Taq DNA sequencing kit from Boehringer Mannheim. After direct blotting onto a membrane (GATC; Konstanz, Germany) and W-crosslinking, the DIG-labeled fragments were detected with anti-dig antibodies conjugated to alkaline phosphatase and chemiluminescent substrate. RESULTS Point mutations in the G-CSF receptor cytoplasmic domain in neutrophils from two patients with congenital neutropenia have been described previously [l 1, 181. In our laboratory, we have investigated 3 patients with severe congenital neutropenia, seven patients with idiopathic neutropenia and six patients with cyclic neutropenia for the frequency of G-CSF receptor point mutations in chronic neutropenia. Additionally, 1 patients with severe aplastic anemia, another preleukemic disorder, were tested for the G-CSF receptor mutation. All patients tested received r-methug-csf at varying doses. The intracellular part of the G-CSF receptor, including the critical region from nucleotide 2384 to 2429, was amplified from cdna and genomic DNA of neutrophils and mononuclear cells using specific primers in a PCR. PCR products were either sequenced directly in both directions or subsequent to cloning into a bluescript vector and transformation in E. coli. Twenty-six of the 3 severe congenital neutropenia patients we analyzed showed a normal G-CSF receptor in the intracellular region from nucleotide position 236 to Four severe congenital neutropenia patients displayed point mutations in this region of the G-CSF receptor gene. The mutations replace a cytosine by a thymine, thereby introducing a stop-codon in the G-CSF receptor mrna (Fig. 1). The G-CSF receptor point mutations were detectable both at the level of cdna and genomic DNA in neutrophils, mononuclear cells and bone marrow cells isolated from the patients. Interestingly, the subgroup of the four congenital neutropenia patients with receptor mutations included the two patients who had developed secondary AML. The two other patients with the mutated G-CSF receptor gene showed no signs of MDS/AML thus far. By analyzing family members of the congenital neutropenia patients with a mutated G-CSF receptor we have shown that the receptor mutations are not inherited [12]. G-CSF receptor analyses at various time points in the life of the patients revealed that the receptor mutations occur spontaneously without correlation to start, duration or dose of G-CSF treatment (Fig. 2). Interestingly, in patient 11 the point mutation could not be detected up to three months before the diagnosis of AML at the age of nine years. In patient 16, who has been previously described in detail [ 111, the mutated G-CSF receptor

4 116 G-CSF Receptor Mutations WT GTT TCC ACC CAG CCC CAA TCC CAG TCT GGC ACC AGC GAT CAG GTC CTT TAT GGG CAG CTG CTG,..... Val Ser Thr Gln Pro Gin Ser Gln Ser Gly Thr Ser Asp Gln Val Leu Tyr Gly Gln Leu Leu * Patients... GTT TCC ACC CAG CCC CAA TCC CAG TCT GGC ACC AGC GAT CAG GTC CTT TAT GGG TAG CTG CTG Val Ser Thr Gln Pro Gln Ser Gln Ser Gly Thr Ser Asp Glii Val Leu Tyr Gly stop patient GTT TCC ACC CAG CCC CAA TCC CAG TCT GGC ACC AGC GAT TAG GTC ClT TAT GGG CAG CTG CTG... * Val Ser Thr Gin Pro Gln Ser Gin Ser Gly Thr Ser Asp stop * 2384 patient... GTT TCC ACC TAG CCC CAA TCC CAG TCT GGC ACC AGC GAT CAG GTC CTT TAT GGG CAG CTG CTG Val Ser Thr stop 2431 Figure 1. Localization of the G-CSF receptorpoint mutations in the G-CSF receptor cdna inpatients with severe congenital neutropenia. The nucleotide and predicted amino acid sequences of the normal wild-type and the mutated G-CSF receptor in four patients with severe congenital neutropenia are shown. The mutated receptor could be detected at the level ofgenomic DNA and mrna in neutrophils and mononuclear cells. Arrows indicate the mutated nucleotides and the numbers represent the nucleotide positions of the G-CSF receptor cdna according to Fukunaga [16]. G-CSF-R monosomy mutation 7* MDS* AML* died AML I I I I Patient 16 I + G-CSF-R normal normal normal mutation G-CSF-R G-CSF-R G-CSF-R, / died Patient 11! 1 I I I normal G-CSF-R Patient 15! G-CSF-R mutation G-CSF-R mutation * * Patient 125 I I I * I w I I I I I I I I I I I I I I I I I I start G-CSF Figure 2. Time course of occurence of G-CSF receptor mutations in severe congenital neutropenia patients undergoing r-methug-csf treatment. Genomic DNA or cdna from neutrophils and mononuclear cells of the four patients with severe congenital neutropenia with a mutated G-CSF receptor gene were analyzed at different time points. The patients received a daily dose of I pg/kg/day (16 and IOS), 3 pg/kg/day (12.5) and 2 pg/kg/day (11). Sequencing of the G-CSF receptor at different time points demonstrated either a normal or mutated (*) G-CSF receptor.

5 Tidow, Pilz, Kasper et al. 117 was detectable before monosomy 7 was diagnosed at the age of 22 years. Patient 15 displayed no G-CSF receptor point mutations in samples taken in 1987, but in myeloid cells from 1994 and 1996 the mutated G-CSF receptor was detectable. In myeloid cells from patient 125, the mutated G-CSF receptor could be detected in samples from 1992 to the present. During the course of treatment with r-methug-csf, the congenital neutropenia patients who acquired the G-CSF receptor mutations showed no changes in their response to treatment with r-methug-csf [12]. The same doses of G-CSF were given before and after the acquisition of the receptor mutation. To summarize, the four congenital neutropenia patients with a mutated G-CSF receptor gene received various doses of r-methug-csf at distinct ages and over different time periods. To determine whether G-CSF receptor point mutations might occur in patients with other forms of chronic neutropenia undergoing G-CSF therapy, we tested six patients with cyclic neutropenia and seven patients with idiopathic neutropenia. These 13 patients showed a normal G-CSF receptor mrna in their myeloid cells. As a control group, we investigated the G-CSF receptor critical region in mononuclear cells from 1 severe aplastic anemia patients undergoing G-CSF treatment; none of the 1 aplastic anemia patients displayed a mutated G-CSF receptor (Table 1). l abk 1. Summary of the patients with chronic neurropenia and aplasiic anemia undergoing G-CSF treatment, and the frequency of the G-CSF receptor mutations Diagnosis Severe congenital neutropenia Cyclic neutropenia Idiopathic neutropenia Severe aplastic anemia Patients tested Mutated G-CSF receptor 4 Total 53 4 DISCUSSION We have demonstrated that point mutations in the critical region of the intracellular part of the G-CSF receptor occur in a subgroup of patients with congenital neutropenia. Four of 3 congenital neutropenia patients displayed a mutated G-CSF receptor, two of whom developed AML. Touw and colleagues [ 191 reported on five of 25 congenital neutropenia patients with mutations in the G-CSF receptor cytoplasmic domain, four of whom developed AML. Taken together, five patients (one patient was analyzed by both groups) with AML secondary to congenital neutropenia with a mutated G-CSF receptor have been described by Touw [19] and our group [12]. Despite the low number of cases, these data might suggest an involvement of the G-CSF receptor mutations in leukemogenesis. In another subgroup of congenital neutropenia patients with MDS/AML, mutations in the rus gene have been reported and linked to leukemogenesis [2]. Interestingly, none of the patients with cyclic neutropenia, idiopathic neutropenia or aplastic anemia showed a mutated G-CSF receptor. These results demonstrate that G-CSF receptor point mutations are not a consequence of G-CSF treatment. Analyses of the family members of the two patients with a mutated G-CSF receptor showed no mutations in the critical G-CSF receptor cytoplasmic domain in myeloid cells from the healthy parents and siblings of patients 16 and 11. Intriguingly, the brother of patient 11 suffers from severe congenital neutropenia too, and he shows a normal G-CSF receptor gene [12]. These data showed that the G-CSF receptor mutation was not inherited and that it might not represent the cause for congenital neutropenia. The G-CSF receptor gene was investigated at different time points in the patients lives. No relationships became apparent between the presence of the G-CSF receptor mutations and the patients age, duration of G-CSF treatment and dose of r-methug-csf. In addition, the time between occurrence of G-CSF receptor

6 118 G-CSF Receptor Mutations c S 3-3 A 8 ii ;i!; I,.., I i! i! i i i: j! I ; ; \ ; I i! i: i! i i 6; i: ii,.. ii ; I :! i! i j I i L Figure 3. Expression of G-CSF receptor on neutrophils from upatient with severe congenitul neutropenia and a healthy control person. G-CSF receptor expression on peripheral blood neutrophils was analyzed by flow cytometry using monoclorial anti-g-csf receptor antibody. In A) neutrophils from patient 125 who suffers from severe congenital neutropeizia and showed a mutated G-CSF receptor gene in sequence analyses and in B) neutrophils from a normal individual were tested. mutations and the development of AML was variable in the affected patients. These data show that G-CSF receptor point mutations are a spontaneous event in the course of neutropenia. In patients with a mutated G-CSF receptor gene, the defective receptor is also expressed at the level of mrna. The mutation is present only in one allele of the G-CSF receptor gene. Therefore, on the protein level this would result in the expression of both forms of the G-CSF receptor protein on the cell surface. In flow cytometry analyses using monoclonal anti-g-csf receptor antibodies, expression of G-CSF receptor protein on neutrophils from patients with a mutated receptor gene could be demonstrated (Fig. 3). However, flow cytometry analyses did not allow discrimination between normal or truncated receptor proteins. In preliminary Western blot analyses of total cell lysates the potentially truncated G-CSF receptor protein could not be detected (B. Kasper, manuscript in preparation). Expression of the normal G-CSF receptor on neutrophils from congenital neutropenia patients with a mutated receptor allele would explain the in vivo unaltered responsiveness of the patients to G-CSF treatment. Possibly, the mutated G-CSF receptor mrna or protein is recognized by control and repair systems and therefore not expressed or degraded. Alternatively, the truncated receptor protein is expressed but the defective signal is overcome by other partners in intracellular signaling. A possible mechanism of leukemogenesis in the patients with a mutated G-CSF receptor gene might be the acquisition of additional mutations in oncogenes or proteins of the control and repair systems. In the latter case, the defective G-CSF receptor protein is no longer eliminated and expression of the truncated receptor on the cell surface would trigger the transduction of a highly proliferative signal. In transfection experiments, introducing the mutated G-CSF receptor gene into murine leukemic cell lines demonstrated that these cells proliferate in response to G-CSF, but do not differentiate like wild-type transfected cells [ 1 I, Functional domains of the G-CSF receptor have been described and the C-terminal part that is truncated by the point mutations has been linked to the transduction of maturation signals [ To conclude, our data argue that point mutations in the critical cytoplasmic region of the G-CSF receptor are spontaneously acquired mutations in a subgroup of severe congenital neutropenia patients. No correlation between G-CSF start, duration and dose of treatment could be seen in our patients. Since patients suffering from other forms of chronic neutropenia (cyclic or idiopahc) and severe aplastic anemia patients display a normal G-CSF receptor, the specific role of receptor point mutations in severe congenital neutropenia, possibly in leukemogenesis, remains to be investigated.

7 Tidow, Pilz, Kasper et al. 1 I9 ACKNOWLEDGMENT This work was supported in part by grant We 942/4-1 from the Deutsche Forschungsgemeinschaft, Bonn, by the Madeleine-Buhler-Kderkrebs-Stiftung, Furth, Germany, and by Amgen Inc., Thousand Oaks, CA. REFERENCES Kostmann RRO. Infantile genetic agranulocytosis (agranulocytosis infantilis hereditaria): a new recessive lethal disease in man. Acta Paediatr Scand 1956;45: Kostmann RRO. Infantile genetic agranulocytosis: a review with presentation of ten new cases. Acta Paediatr Scand 1975;64: Bonilla MA, Gillio AP, Ruggeiro M et al. Effects of recombinant human granulocyte colony-stimulating factor on neutropenia in patients with congenital agranulocytosis. N Engl J Med 1989;32: Welte K, Zeidler C, Reiter A et al. Differential effects of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in children with severe congenital neutropenia. Blood 199;75: Dale DC, Bonilla MA, Davis MW et al. A randomized controlled phase-111 trial of recombinant human granulocyte colony-stimulating factor (Filgrastim) for treatment of severe chronic neutropenia. Blood 1993;81: Bonilla MA, Dale D, Zeidler C et al. Long-term safety of treatment with recombinant human granulocyte colony-stimulating factor (r-methug-csf) in patients with severe congenital neutropenia. Br J Haematol 1994;88: Welte K, Dale D. Pathophysiology and treatment of severe chronic neutropenia. Ann Hematol 1996;72: Welte K, Boxer LA. Severe chronic neutropenia: pathophysiology and therapy. Semin Hematol 1997 (in press). Rosen R, Kang S. Congenital agranulocytosis terminating in acute myelomonocytic leukemia. J Pediatr 1979;94: Rauprich P, Kasper B, Tidow N et al. The protein tyrosine kinase JAK2 is activated in neutrophils from patients with severe congenital neutropenia. Blood 1995;86: Dong F, Brynes RK, Tidow Net al. 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: 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: Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal Biochem 1987;162: Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol 1987;155; Chien A, Edgar DB, Trela JM. Deoxynbonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 1976;127: Fukunaga R, Seto Y, Mizushima S et al. Three different mrnas encoding human granulocyte colony-stimulating factor receptor. Proc Natl Acad Sci USA 199;87: Sanger F, Miklen S, Coulson AR. DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 1977:74: Dong F, Hoefsloot LH, Schelen AM 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; Touw IP, de Koning JP, Lowenberg B et al. Defective G-CSF receptors in severe congenital neutropenia (SCN) associated with progression towards MDS and AML. Blood 1995;86(suppl 1):26a. 2 Kalra R, Dale D, Freedman M et al. Monosomy 7 and activating RAS mutations accompany nialignant transformation in patients with congenital neutropenia. Blood 1995;86: Fukunaga R, Ishizaka-Ikeda E, Pan CX et al. Functional domains of the granulocyte colony-stimulating factor receptor. EMBO J 1991 ;1: Fukunaga R, Ishzaka-keda E, Nagata S. Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stitnulating factor receptor. Cell 1993;74: Dong F, van Buitenen C, Pouwels K et al. Distinct cytoplasmic regions of the human G-CSF receptor involved in transduction of proliferative and maturation signals. Mol Cell Biol 1993;13: ,

8 12 G-CSF Receptor Mutations DISCUSSION Dr. Fibbe: Do you think that all patients with the mutated receptor will ultimately get leukemia? Dr. Welte: I do not know for sure. However, our hypothesis is that all patients who acquire the mutation also develop leukemia. We have started to collect bone marrow and peripheral blood progenitor cells in these patients as backup in case they develop leukemia because if they develop leukemia, treatment outcome is very poor. None of the patients with chemotherapy and only few patients who underwent bone marrow transplantation survived. Dr. Lowenberg: Would you consider doing an allogeneic transplant? Dr. Welte: We have performed allogeneic transplantations using bone marrow from HLA-identical siblings in a subgroup of ahout 2%-3% who do not respond to G-CSF. All of them survived. Dr. Ihle: In those patients that develop AML, are the AML blast cells responsive to G-CSF, and what happens when you remove G-CSF from those patients? Dr. Welte: In one patient who developed AML, we stopped G-CSF immediately. She had about 7, blastslpl in peripheral blood, which decreased gradually to about 1, within four days after discontinuation of G-CSF. But then she didn t decrease her blast cell counts further. Subsequently, the malignant clone expanded again by itself, and the patient developed fever, thrombocytopenia and other leukemic-associated symptoms. Therefore, we had to initiate the anti-leukemic treatment. The patient s blasts had receptors for G-CSF and responded to G-CSF in vitro. Dr. Ihle: Do they retain the absolute dependence on G-CSF or growth factors in vitro? Dr. Welte: No