Phil J. Ancliff

Notch-1 Mutations Are Secondary Events in Some Patients with T-Cell Acute Lymphoblastic Leukemia

Purpose: Activating Notch-1 mutations are frequent in T-cell acute lymphoblastic leukemia (T-ALL), occurring in >50% of patients. In murine models of T-ALL, Notch-1 activation can both directly initiate leukemia and cooperate secondarily to other primary events. Whether acquisition of Notch-1 mutations is an early initiating event or a secondary event in the pathogenesis of human T-ALL is unclear. Experimental Design: We used denaturing high-performance liquid chromatography, sequencing, and fragment analysis to analyze Notch-1 mutational status and mutant level in 62 patients at presentation as well as 16 matched presentation-relapse samples. Results: We detected Notch-1 mutations in 47 patients (76%). Seven of these were low-level mutations (quantified at ≤10%), despite high blast counts, suggesting that they were acquired as a secondary event in a subclone. Of 16 matched presentation-relapse samples studied, 7 were wild-type at both presentation and relapse. Five of nine mutant-positive patients at presentation relapsed with the same mutation(s) at the same high level. Four patients had evidence of a change in mutant at relapse. One lost a PEST mutation and became wild-type. Two others lost mutations at relapse but acquired different mutations, despite unchanged T-cell receptor rearrangements, suggesting that the latter event predated the acquisition of the Notch-1 mutation. One relapsed with a secondary T-cell leukemia and different Notch mutation. Conclusions: These results suggest that Notch-1 mutations can sometimes be acquired as secondary events in leukemogenesis and must be used cautiously as solitary minimal residual disease markers.

Long-term follow-up of granulocyte colony-stimulating factor receptor mutations in patients with severe congenital neutropenia: implications for leukaemogenesis and therapy

Summary. Severe congenital neutropenia (SCN) is characterized by profound neutropenia, recurrent severe bacterial infections and maturation arrest in the myeloid lineage. Granulocyte colony‐stimulating factor (G‐CSF) treatment results in clinical improvement in over 90% of cases. Point mutations of the G‐CSF receptor (G‐CSFR) have been implicated in the progression of SCN to acute myeloid leukaemia (AML). Data are presented here on the 9‐year follow‐up of seven patients and the further screening of 18 other cases. One of the two original cases with a G‐CSFR mutation has improved clinically; nevertheless, mutant DNA could still be detected at a very low level > 8 years after identification. The second child with a mutation progressed to myelodysplasia/AML 5 years after her mutation was detected. No mutations were found in the 18 new cases. One of three transformed cases had a G‐CSFR mutation. This work is in agreement with the suggestion that G‐CSFR mutations may provide a survival advantage to haemopoietic stem cells, but argues against the inevitability of leukaemic progression in their presence. Furthermore, the low frequency of G‐CSFR mutations in SCN and the importance of regular screening and close clinical and laboratory follow‐up if a mutation is found were demonstrated.

Recent advances in the understanding of genetic defects of neutrophil number and function

Neutrophils are amongst the first immune cells to arrive at sites of infection and play an important role as the host’s first line of defence against invading pathogens. Defects of neutrophil number or function are usually recognized clinically by recurrent infections that often are life‐threatening. Over the last few years, a number of genetic mutations have been discovered to be the basis for congenital neutropenia, adding to our understanding of the molecular basis of these diseases. While many genetic mutations that cause severe congenital neutropenia result in a differentiation block at the promyelocyte stage, defects of neutrophil function are more heterogeneous on clinical, genetic and mechanistic levels. In this review we discuss recent advances in our understanding of the genetic and molecular basis of human neutrophil disorders.

GATA2 mutations in sporadic and familial acute myeloid leukaemia patients with CEBPA mutations.

GATA2 mutations have recently been reported in acute myeloid leukaemia (AML) patients with CEBPA‐double mutations. To explore their impact on this favourable‐risk disease, we determined GATA2 status in 153 sporadic AML patients and three members of a germ‐line CEBPA‐mutant family at AML presentation. Overall, 27% (15/55) CEBPA‐double, 16% (7/43) CEBPA‐single and 0% (0/55) normal karyotype/CEBPA‐wild‐type patients were GATA2‐mutant. All familial AML patients acquired both a second CEBPA and a GATA2 mutation. CEBPA and GATA2 mutant levels indicated that both mutations were likely to be early events in leukaemogenesis. GATA2 status did not impact on the favourable outcome of CEBPA‐double/FLT3‐inernal tandem duplication‐negative patients.

Homozygous HAX1 mutations in severe congenital neutropenia patients with sporadic disease: a novel mutation in two unrelated British kindreds

Patients with autosomal dominant (AD), sporadic and X‐linked severe congenital neutropenia (SCN) may have mutations in the elastase 2 (ELA2) or Wiskott‐Aldrich syndrome (WAS) genes. Homozygous mutations in the HAX1 gene have recently been reported in autosomal recessive (AR) cases of primarily Middle‐Eastern descent and the original Kostmann family. We screened 109 predominantly Caucasian SCN kindreds for mutations in these genes; 33 (30%) had 24 different ELA2 mutations, five of them novel, two kindreds (2%) had WAS mutations and four kindreds (4%) had three different HAX1 mutations, two of them novel. One HAX1 mutation (p.Ser43LeufsX11) was found in an AR Ashkenazi Jewish kindred, the other (p.Glu31LysfsX54) in two unrelated British patients with sporadic disease. Microsatellite analysis of the HAX1 locus revealed a common haplotype (maximum distance 4·1 Megabases) for the p.Glu31LysfsX54 patients, suggesting a possible ancestral founder. In functional assays, the level of spontaneous and staurosporine‐induced apoptosis was increased in neutrophils from both p.Ser43LeufsX11 patients but not a p.Glu31LysfsX54 patient, suggesting the possible presence of modifying factors. The low incidence of HAX1 mutations in our study suggests that the frequency may vary between racial groups but suggests that irrespective of inheritance or racial origin, SCN patients should be screened for HAX1 mutations.

Stem cell transplantation in severe congenital neutropenia: an analysis from the European Society for Blood and Marrow Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment of severe congenital neutropenia (SCN), but data on outcome are scarce. We report on the outcome of 136 SCN patients who underwent HSCT between 1990 and 2012 in European and Middle East centers. The 3-year overall survival (OS) was 82%, and transplant-related mortality (TRM) was 17%. In multivariate analysis, transplants performed under the age of 10 years, in recent years, and from HLA-matched related or unrelated donors were associated with a significantly better OS. Frequency of graft failure was 10%. Cumulative incidence (day +90) of acute graft-versus-host disease (GVHD) grade 2-4 was 21%. In multivariate analysis, HLA-matched related donor and prophylaxis with cyclosporine A and methotrexate were associated with lower occurrence of acute GVHD. Cumulative incidence (1 year) of chronic GVHD was 20%. No secondary malignancies occurred after a median follow-up of 4.6 years. These data show that the outcome of HSCT for SCN from HLA-matched donors, performed in recent years, in patients younger than 10 years is acceptable. Nevertheless, given the TRM, a careful selection of HSCT candidates should be undertaken.

Phenotypic heterogeneity and evidence of a founder effect associated with G6PC3 mutations in patients with severe congenital neutropenia

Causative homozygous or compound heterozygous mutations in the glucose-6-phosphatase, catalytic subunit 3 gene (G6PC3) have recently been described for a sub-group of patients with severe congenital neutropenia (SCN) (Arostegui et al, 2009; Boztug et al, 2009, 2011; Xia et al, 2009; Germeshausen et al, 2010; Banka et al, 2011a,b; Hayee et al, 2011). In addition to neutropenia and recurrent infections, mutant-positive patients are reported to have varied other congenital abnormalities, such as a prominent superficial venous pattern, structural heart defects, urogenital malformations, skeletal abnormalities, primary pulmonary hypertension, growth and developmental delay (Boztug et al, 2011). Several reports have also indicated considerable variation in bone marrow (BM) morphology, including a lack of the myeloid maturation arrest characteristic of SCN (McDermott et al, 2010; Banka et al, 2011b). We screened 108 CN patients or kindreds, known wild-type for the ELANE, HAX1 and WAS genes, for G6PC3 mutations and identified further phenotypic heterogeneity, in that two mutant-positive patients had neutropenia alone. Another presented with isolated neutropenia and was found to have clinically asymptomatic cardiac abnormalities. The study had Local Regional Ethical Committee approval. Informed consent was obtained from patients and/or their parents. Polymerase chain reaction (PCR) amplicons of the six G6PC3 exons were screened using denaturing high performance liquid chromatography (Transgenomic Limited, Glasgow, UK) (see Table S1). Samples with abnormal chromatograms were sequenced. Mutations were confirmed by PCR with mismatch primers and restriction enzyme digestion (Table S2). Three different homozygous G6PC3 mutations were detected in four of the 108 patients/kindreds studied. Parents of affected individuals were all heterozygous for the mutation. Patient 1 (born 1982, from Pakistan, parents first cousins) had a homozygous 21 bp deletion in exon 1 (c.190_210del) resulting in an in-frame deletion of seven amino acids in the second trans-membrane domain (p.Thr64_Ile70del) (Fig 1A, B). The patients’ phenotype was similar to that of the G6PC3-mutated patients described by Boztug et al (2009). In addition to neutropenia (absolute neutrophil count [ANC] 0·1 × 109/l), first noted aged 9 years, he suffered from recurrent oral ulcers, aspergillus infection and recurrent episodic bacterial pneumonia, but responded to standard antibiotic therapy and now receives regular granulocyte colony-stimulating factor (G-CSF). He was born with an atrial septal defect and has granulomatous inflammatory bowel disease, splenomegaly, digital clubbing and short stature. His affected sister is also homozygous G6PC3 mutant and has a similar phenotype but without the atrial septal defect and opportunistic infection. Fig 1 WAVE chromatograms, restriction enzyme digests and haplotype study of G6PC3 mutant-positive kindreds. (A) WAVE chromatograms of exon 1 and exon 5 polymerase chain reaction (PCR) products from mutation-positive patients mixed with known wild-type (WT) ... Patient 2 (born 2001, from Turkey, consanguineous parents) has a novel c.623T>G mutation in exon 5 resulting in p.Leu208Arg (Fig 1A, B). She presented in infancy with severe neutropenia (ANC <0·1 × 109/l), recurrent otitis media, chronic gingivitis and periodontitis. Initially responsive to 5 μg/kg G-CSF, subsequent doses in excess of 10–15 μg/kg have failed to control her infections. Although otherwise clinically asymptomatic and with no obvious congenital anomalies, echocardiography post-G6PC3 mutation detection showed a patent foramen ovale and tricuspid insufficiency. Patients 3 and 4, both from Pakistan, are unrelated but have the same homozygous missense mutation in exon 1, c.130C>T resulting in p.Pro44Ser (Fig 1A, B). Both have normal echocardiograms, lack other congenital abnormalities and have had normal growth and development. Patient 3 (born 1989, non-consanguineous parents) presented with neutropenia at age 13 years with intermittent mouth ulcers (ANC 0·4 × 109/l) and has suffered a single bout of myositis. His only treatment has been intermittent G-CSF for recurring severe mouth ulcers. Patient 4 (born 2002, parents first cousins) presented at 3 years of age with recurrent upper respiratory and chest infections, and has had two instances of gluteal abscesses. He has otherwise been well, with a recent ANC of 0·45 × 109/l, and has not received G-CSF. Of note, both patients have normocellular BM morphology with normal neutrophils, although slightly left-shifted myelopoiesis was reported for Patient 3. The mutation was not detected in samples from 54 haematologically normal individuals of Pakistani origin but was reported recently in an SCN patient, also of Pakistani origin, who similarly had normal BM cellularity without maturation arrest, although further information on the patient was not available (Banka et al, 2011b). Pro44 and Leu208 are both conserved amongst species (Fig S1) and, due to their trans-membranous location, the mutations are predicted to be disruptive to normal G6PC3 function. A possible ancestral founder for the p.Pro44Ser mutation was identified from a common haplotype consisting of two highly polymorphic micro-satellite markers and four single nucleotide polymorphisms spanning a maximum of 4·8Mb in the two unrelated p.Pro44Ser-mutated patients (Fig 1C) (PCR primers and conditions in Table S3). None of 20 Pakistani control samples carried the entire six-marker haplotype, and the probability of this conserved haplotype homozygously presenting in the Pakistani population would be very low. A functional impact on apoptosis consistent with a pathogenic mutation was demonstrated for purified neutrophils from Patients 1 and 4 using previously published assays (Smith et al, 2009). Both patients had a high level of spontaneous apoptosis (26·4%, 38·9% non-viable cells respectively at time zero compared to 3·6 ± 4·7% [mean±2SD] for 12 normal controls) (Fig 2). After 3 h incubation this level had significantly increased in Patient 1 compared to controls (58·2% versus 10·5 ± 7·1%) and was further enhanced in the presence of staurosporine (92·8% versus 12·9% ± 9·5% at 3 h). For Patient 4, the level of apoptosis only increased in the presence of staurosporine (36·4% without, 69·5% with staurosporine at 3 h). In addition, severe neutrophil glycosylation defects have been reported for Patients 1 and 3 (Hayee et al, 2011). Fig 2 Percentage of non-viable neutrophils in patients with the p.Thr64_Ile70del and p.Pro44Ser mutations, their carrier parents and controls. Graphical representation of the percentage of non-viable neutrophils (Annexin V-positive, propidium iodide-negative ... Overall, the frequency of G6PC3 mutations in our SCN cohort was 3% (4 of 155 patients/kindreds), although this incidence is likely to depend on the racial composition of the patient population investigated. No evidence has been found for a G6PC3 genotype-phenotype correlation (Banka et al, 2011b; Boztug et al, 2011), and it has been suggested that different founder mutations may exist according to racial groups (Banka et al, 2011b). Whether such variable genetic inheritance patterns has a wider impact on phenotype will require analysis of many cases, and the underlying reasons for the phenotypic heterogeneity associated with G6PC3 mutations are therefore currently unclear. Nevertheless, based on our observations of milder disease, isolated neutropenia and normal BM morphology but evident functional abnormalities in our two p.Pro44Ser-mutated cases, we would recommend G6PC3 screening as part of the work-up of all CN/SCN patients lacking other mutations, irrespective of the presence of additional congenital abnormalities.

MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia

Heterozygous mutations in MECOM (MDS1 and EVI1 complex locus) have been reported to be causative of a rare association of congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Here we report on 12 patients with congenital hypomegakaryocytic thrombocytopenia caused by MECOM mutations (including 10 novel mutations). The mutations affected different functional domains of the EVI1 protein. The spectrum of phenotypes was much broader than initially reported for the first 3 patients; we found familial as well as sporadic cases, and the clinical spectrum ranged from isolated radioulnar synostosis with no or mild hematological involvement to severe bone marrow failure without obvious skeletal abnormality. The clinical picture included radioulnar synostosis, bone marrow failure, clinodactyly, cardiac and renal malformations, B-cell deficiency, and presenile hearing loss. No single clinical manifestation was detected in all patients affected by MECOM mutations. Radioulnar synostosis and B-cell deficiency were observed only in patients with mutations affecting a short region in the C-terminal zinc finger domain of EVI1. We propose the term MECOM-associated syndrome for this heterogeneous hereditary disease and inclusion of MECOM sequencing in the diagnostic workup of congenital bone marrow failure.

Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Moulding et al. 2007. J. Exp. Med. doi:10.1084/jem.20062324 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20062324%26rft_id%253Dinfo%253Apmid%252F17724125%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%