Truncated CSF3 receptors induce pro-inflammatory responses in severe congenital neutropenia

Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony-stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient-derived CSF3R-truncating mutation in ELANE-SCN and HAX1-SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R-mutant control HPCs, CSF3R-mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN-HPCs does not evoke clonal outgrowth but causes a sustained pro-inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN.     

Distinct patterns of mutations occurring in de novo AML versus AML arising in the setting of severe congenital neutropenia

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Like most other bone marrow failure syndromes, it is associated with a marked propensity to transform into a myelodysplastic syndrome (MDS) or acute leukemia, with a cumulative rate of transformation to MDS/leukemia that exceeds 20%. The genetic (and/or epigenetic) changes that contribute to malignant transformation in SCN are largely unknown. In this study, we performed mutational profiling of 14 genes previously implicated in leukemogenesis using 14 MDS/leukemia samples from patients with SCN. We used high-throughput exon-based resequencing of whole-genome-amplified genomic DNA with a semiautomated method to detect mutations. The sensitivity and specificity of the sequencing pipeline was validated by determining the frequency of mutations in these 14 genes using 188 de novo AML samples. As expected, mutations of tyrosine kinase genes (FLT3, KIT, and JAK2) were common in de novo AML, with a cumulative frequency of 30%. In contrast, no mutations in these genes were detected in the SCN samples; instead, mutations of CSF3R, encoding the G-CSF receptor, were common. These data support the hypothesis that mutations of CSF3R may provide the "activated tyrosine kinase signal" that is thought to be important for leukemogenesis.     

Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia

Severe congenital neutropenia (SCN) is a BM failure syndrome with a high risk of progression to acute myeloid leukemia (AML). The underlying genetic changes involved in SCN evolution to AML are largely unknown. We obtained serial hematopoietic samples from an SCN patient who developed AML 17 years after the initiation of G-CSF treatment. Next- generation sequencing was performed to identify mutations during disease progression. In the AML phase, we found 12 acquired nonsynonymous mutations. Three of these, in CSF3R, LLGL2, and ZC3H18, co-occurred in a subpopulation of progenitor cells already in the early SCN phase. This population expanded over time, whereas clones harboring only CSF3R mutations disappeared from the BM. The other 9 mutations were only apparent in the AML cells and affected known AML-associated genes (RUNX1 and ASXL1) and chromatin remodelers (SUZ12 and EP300). In addition, a novel CSF3R mutation that conferred autonomous proliferation to myeloid progenitors was found. We conclude that progression from SCN to AML is a multistep process, with distinct mutations arising early during the SCN phase and others later in AML development. The sequential gain of 2 CSF3R mutations implicates abnormal G-CSF signaling as a driver of leukemic transformation in this case of SCN.     

Risk of myelodysplastic syndrome and acute myeloid leukemia in congenital neutropenias

Granulocyte colony-stimulating factor (G-CSF) has had a major impact on the management of "severe chronic neutropenia" (SCN), a collective term referring to congenital, idiopathic, or cyclic neutropenia. Almost all patients respond to G-CSF with increased neutrophils, reduced infections, and improved survival. Some responders with congenital neutropenia and Shwachman-Diamond syndrome (SDS) have developed myelodysplastic syndrome and acute myeloid leukemia (MDS/AML), which raises the question of the role of G-CSF in pathogenesis. The issue is complicated because both disorders have a propensity for MDS or AML as part of their natural history. To address this, the Severe Chronic Neutropenia International Registry (SCNIR) used its large database of chronic neutropenia patients treated with G-CSF to determine the incidence of malignant myeloid transformation in the two disorders, and its relationship to treatment and to other patient characteristics. No statistically significant relationships were found between age at onset of MDS or AML and patient gender, G-CSF dose, or duration of G-CSF therapy. What was observed, however, was the multistep acquisition of aberrant cellular genetic changes in marrow cells from patients who transformed, including activating ras oncogene mutations, clonal cytogenetic abnormalities, and G-CSF receptor mutations. In murine models, the latter produces a hyperproliferative response to G-CSF, confers resistance to apoptosis, and enhances cell survival. Since congenital neutropenia and SDS are inherited forms of bone marrow failure, G-CSF may accelerate the propensity for MDS/AML in the genetically altered stem and progenitor cells, especially in those with G-CSF receptor and ras mutations (82% and 50% of patients who transform, respectively). Alternatively, and equally plausible, G-CSF may simply be an "innocent bystander" that corrects neutropenia, prolongs patient survival, and allows time for the malignant predisposition to declare itself. In patients who transform to overt MDS or AML, hematopoietic stem cell transplantation is the only chance for cure. In those with "soft" signs of MDS, such as an isolated clonal cytogenetic change but without other evidence of MDS, or with an isolated G-CSF receptor mutation, there is room for conservative management. One option is to reduce the G-CSF dosage as much as possible, and observe the tempo of progression, if any, to more overt signs of malignancy.     

G-CSF receptor (CSF3R) mutations in X-linked neutropenia evolving to acute myeloid leukemia or myelodysplasia

X-linked neutropenia (XLN) is a rare form of Congenital Neutropenia (CN) caused by inherited gain-of-function mutations of WAS. Here we report 2 cases of the original L270P X-linked neutropenia kindred that evolved to MDS or AML, with acquisition of G-CSFR (CSF3R) mutations and monosomy 7. Thus, leukemic transformation with acquisition of CSF3R mutations and monosomy 7 is not restricted to classical congenital neutropenia with autosomal inheritance, but can also occur in other genotypes of inherited neutropenia.     

Two case studies of chronic idiopathic neutropenia preceding acute myeloid leukaemia

Chronic idiopathic neutropenia is regarded as a benign disorder without risk of malignant transformation. We present two patients with chronic idiopathic neutropenia who showed disease progression to acute myeloid leukaemia. Sequence analysis of the granulocyte-colony stimulating factor receptor (G-CSFR) gene from leukaemic DNA did not reveal any mutations and microsatellite analysis provided no evidence of microsatellite instability or loss of constitutional heterozygosity. These case studies suggest that chronic idiopathic neutropenia may constitute a preleukaemic condition in some patients. Alterations of the G-CSFR or defective DNA mismatch repair do not appear to be involved in malignant transformation.     

Clinical implications of ELA2-, HAX1-, and G-CSF-receptor (CSF3R) mutations in severe congenital neutropenia

Congenital Neutropenia (CN) is a heterogeneous bone marrow failure syndrome characterized by a maturation arrest of myelopoiesis at the level of the promyelocyte/myelocyte stage with peripheral blood absolute neutrophil counts below 0·5 × 10⁹/l. There are two major subtypes of CN as judged by inheritance: an autosomal dominant subtype, e.g. defined by neutrophil elastase mutations (approximately 60% of patients) and an autosomal recessive subtype (approximately 30% of patients), both presenting with the same clinical and morphological phenotype. Different mutations have been described (e.g. HAX1 , p14 etc) in autosomal recessive CN, with HAX1 mutations in the majority of these patients. CN in common is considered as a preleukemic syndrome, since the cumulative incidence for leukemia is more than 25% after 20 years of observation. Leukemias occur in both, the autosomal dominant and recessive subtypes of CN. The individual risk for each genetic subtype needs to be further evaluated. Numbers of patients tested for the underlying genetic defect are still limited. Acquired G-CSFR ( CSF3R ) mutations are detected in approximately 80% of CN patients who developed acute myeloid leukemia independent of the ELA2 or HAX1 genetic subtype, suggesting that these mutations are involved in leukemogenesis. As the majority of patients benefit from G-CSF administration, HSCT should be restricted to non-responders and patients with leukaemic transformation.     

Monosomy 7 and activating RAS mutations accompany malignant transformation in patients with congenital neutropenia

Individuals with severe forms of congenital neutropenia suffer from recurrent infections. The therapeutic use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to increase the neutrophil count is associated with fewer infections and an improved quality of life. However, the long-term effects of this new therapy are largely unknown. In particular, it is unclear if myeloid leukemia, a known complication of some forms of congenital neutropenia, will occur with increased frequency among patients who receive long-term treatment with hematopoietic growth factors. We report 13 patients with congenital disorders of myelopoiesis who developed leukemic transformation with either myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) and 1 who acquired a clonal cytogenetic abnormality without evidence of MDS or AML while receiving rhG-CSF. The bone marrows of 10 patients showed monosomy 7 and 5 had activating RAS mutations. These abnormalities were not detected in pretreatment bone marrows and cessation of rhG-CSF was not associated with either clinical improvement or cytogenetic remission. We conclude that patients with severe forms of congenital neutropenia are at relatively high risk of developing MDS and AML. The occurrence of monosomy 7 and RAS mutations in these cases suggests that the myeloid progenitors of some patients are genetically predisposed to malignant transformation. The relationship between therapeutic rhG-CSF and leukemogenesis in patients with severe chronic neutropenia is unclear.     

Screening for mutation R882 in the DNMT3A gene in Chinese patients with hematological disease

The DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A) gene is actively involved in epigenetic regulation. Mutations in this gene affect disease progression and response to therapy, particularly in hematological disease. Recent studies have demonstrated that DNMT3A gene mutations affecting codon R882 are actively involved in acute myelogenous leukemia (AML), and cause abnormal alteration of DNA methylation and poor survival. In this study, we screened DNMT3A mutations in a total of 389 Chinese patients with hematological disease by sequencing the coding region of exon 23 covering residue R882. Three heterozygous mutations (p.R882S, p.R882C and p.R882H) were identified in three of 61 AML patients, whereas none of patients with other hematological disorders harbored any mutation. Our results support the notion that DNMT3A R882 was a frequent mutation in AML patients but rare in other types of hematological disorder.     

Kostmann syndrome and severe congenital neutropenia

Congenital neutropenia (CN) includes hematologic disorders characterized by severe neutropenia with an absolute neutrophil count (ANC) below 0.5 x 10(9)/L associated with severe systemic bacterial infections from early infancy. One subtype of CN, Kostmann syndrome, was originally described as an autosomal-recessive disorder, characterized by early-stage maturation arrest of myelopoiesis. Autosomal-dominant and sporadic cases have also been reported. Recent studies on the genetic bases of CN have detected different inherited or spontaneous point mutations in the neutrophil elastase gene. Development of additional genetic defects during the course of disease, such as granulocyte colony-stimulating factor (G-CSF)-receptor gene mutations and cytogenetic aberrations, indicates an underlying genetic instability. Data on more than 300 patients with CN collected by the Severe Chronic Neutropenia International Registry (SCNIR) since 1994 demonstrate that, independent of the CN subtype, more than 90% of patients respond to recombinant human (rHu)G-CSF with ANCs that can be maintained at approximately 1.0 x 10(9)/L. Adverse events include mild splenomegaly, moderate thrombocytopenia, osteoporosis, and malignant transformation into myelodysplasia (MDS)/leukemia. If and how rHuG-CSF treatment impacts on these adverse events remains unclear since there are no historical controls for comparison. Hematopoietic stem cell transplantation (HSCT) is still the only available treatment for patients refractory to rHuG-CSF treatment.     

N-RAS gene mutation in patients with aplastic anemia and aplastic anemia/ paroxysmal nocturnal hemoglobinuria during evolution to clonal disease

Long-term survivors of aplastic anemia (AA) have a high incidence of clonal disorders, in particular paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes (MDS), and acute nonlymphocytic leukemia. To investigate the potential involvement of N-RAS gene mutations in the predisposition to leukemic evolution, a subset of patients at potentially increased risk for clonal disease was selected based on evidence of existing clonal evolution. Nine patients showed a monoclonal pattern of X-chromosome inactivation, 18 demonstrated a PNH clone, and in 3 MDS developed during the course of this study. No mutations were detected during the aplastic phase of disease; 2 of 3 patients with MDS after AA also showed no mutations. However, in 1 patient in whom the disease transformed from AA/PNH to MDS, a mutation of GGT --> GAT at N-RAS codon 13 became detectable, whereas the PNH mutation disappeared. The authors conclude that N-RAS mutations are not an early event preceding transformation of AA or AA/PNH to leukemia. In a subset of patients, RAS mutations may occur at the time of evolution to MDS, but preexisting RAS mutations do not explain the propensity of AA to leukemogenesis. Although PNH is also associated with leukemia, this may arise in the non-PNH cells, indicating that PIG-A gene mutation is not per se oncogenic. (Blood. 2000;95:646-650)     

Acute myeloid/NK precursor cell leukemia with trisomy 4 and a novel point mutation in the extracellular domain of the G-CSF receptor in a patient with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) has been well recognized as a granulocytic disorder not associated with increased risk to malignant transformation. Four cases, however, of acute myeloid leukemia have been recently reported in patients with CIN. In the current paper, we report on a CIN patient who developed acute myeloid/natural killer (NK) precursor cell leukemia 11 years after diagnosis and 4 months after initiation of treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Leukemic cells had trisomy 4 as the sole cytogenetic abnormality and, also, a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSFR) leading to truncated protein with a loss of 36 amino acids. There was no evidence that this receptor transmitted signals even in the presence of high doses of rhG-CSF in the cultures. We consider that CIN may be a preleukemic condition, at least in a subset of patients, and that rhG-CSF administration is unlikely to be involved in the leukemic transformation in this patient, although such a possibility could not be completely ruled out.     

Clonal diseases of large granular lymphocytes [see comments]

Three distinct clinical syndromes occur in patients with increased numbers of circulating LGL. Patients with T-LGL leukemia have clonal proliferations of CD3+ LGL typically associated with chronic neutropenia and autoimmune features. NK-LGL leukemia is characterized by clonal CD3- LGL proliferation with an acute clinical presentation marked by massive hepatosplenomegaly and systemic illness. However, most patients with increased numbers of CD3- LGL do not have clinical features of NK-LGL leukemia and have a chronic clinical course. X- linked gene analyses have supported a polyclonal LGL lymphocytosis in this syndrome. Further studies are needed to determine whether clonal progression can occur in these patients.     

Time course of increasing numbers of mutations in the granulocyte colony-stimulating factor receptor gene in a patient with congenital neutropenia who developed leukemia

Point mutations in the granulocyte colony-stimulating factor receptor (G-CSFR) gene have been linked to the development of secondary leukemia in patients with congenital neutropenia (CN). This report presents data on a now 18-year-old patient with CN who has received G-CSF treatment since 1989 and who developed acute myeloid leukemia (AML) in 1998. To evaluate whether there is an association between the occurrence of point mutations of the G-CSFR gene and development of secondary AML, DNA/messenger RNA of neutrophils and mononuclear cells from this patient were analyzed at different time points by polymerase chain reaction and subsequent cloning by DNA sequencing of representative numbers of individual clones. Findings suggest an increasing instability of the G-CSFR gene in time as judged by increasing numbers of mutations proposed to be one important step in the development of AML in this patient.     

HAX1 mutation positive children presenting with haemophagocytic lymphohistiocytosis

The genetic basis of haemophagocytic lymphohistiocytosis (HLH) has not been elucidated in 10% of affected patients. In this study, we report four HLH episodes in three patients with HAX1 gene mutations. We screened the mutations associated with congenital neutropenia (CN) because the neutropenia persisted following HLH treatment. There were homozygous HAX1 mutations detected in all patients. This is the first case series of patients with CN caused by HAX1 mutation who presented with HLH. We hypothesize that severe neutropenia persists after an HLH episode in children without HLH mutations (especially infants) because these patients have CN caused by HAX1 mutations.     

Severe congenital neutropenia

Severe congenital neutropenia (CN) includes a variety of hematologic disorders characterized by severe neutropenia, with absolute neutrophil counts (ANC) below 0.5 x 10(9)/L, and associated with severe systemic bacterial infections from early infancy. One subtype of CN, Kostmann syndrome, is an autosomal recessive disorder, characterized histopathologically by early-stage maturation arrest of myeloid differentiation. CN with similar clinical features occurs as an autosomal dominant disorder and many sporadic cases also have been reported. This genetic heterogeneity suggests that several pathophysiological mechanisms may lead to this common clinical phenotype. Recent studies on the genetic bases of CN have detected inherited or spontaneous point mutations in the neutrophil elastase gene (ELA 2) in about 60% to 80% of patients and, less commonly, mutations in other genes. Acquisition of additional genetic defects during the course of the disease, for example, granulocyte colony-stimulating factor (G-CSF) receptor gene mutations and cytogenetic aberrations, indicates an underlying genetic instability as a common feature for all congenital neutropenia subtypes. Data on more than 600 patients with CN collected by the Severe Chronic Neutropenia International Registry (SCNIR) demonstrate that, regardless of the particular CN subtype, more than 95% of these patients respond to recombinant human (rHu)G-CSF with ANCs that can be maintained above 1.0 x 10(9)/L. Adverse events include mild splenomegaly, osteoporosis, and malignant transformation into myelodysplasia (MDS)/leukemia. If and how G-CSF treatment impacts on these adverse events is not fully understood. In recent analyses the influence of the G-CSF dose required to achieve neutrophil response (ANC >1,000/microL) in the risk of developing acute myeloid leukemia (AML) has been reported. Hematopoietic stem cell transplantation (HSCT) is still the only treatment available for patients who are refractory to G-CSF treatment.     

Mutations in the ELA2 gene correlate with more severe expression of neutropenia: a study of 81 patients from the French Neutropenia Register

Heterozygous mutations of the gene encoding neutrophil elastase (ELA2) have been associated with cyclic neutropenia (CN) and severe congenital neutropenia (SCN). To date, 30 different mutations have been reported, but no correlation has been found with the degree of neutropenia. To address this issue, we analyzed the clinical, hematologic, and molecular characteristics of 81 unrelated patients with SCN (n = 54) or CN (n = 27). We identified mutations in 31 patients, two thirds of whom had sporadic forms. Familial cases were consistent with dominant inheritance. Seventeen novel mutations were identified, showing that the mutational spectrum encompasses not only the region encoding the mature enzyme but also the prodomains and promoter region. Genotype-phenotype analysis strongly suggested that ELA2 mutations correlate with more severe expression of neutropenia, specifically in patients diagnosed with SCN. This study underlines the importance of ELA2 molecular screening to identify patients who may be at particular risk of severe bacterial infections and/or acute myeloid leukemia/myelodysplasia. By phenotypic analysis of affected relatives and carriers of the same ELA2 mutations, we showed that the expression of neutropenia in CN and SCN may be either homogeneous or variable according to the type of mutations, suggesting different pathogenetic mechanisms.