Georgina W. Hall

Guideline for investigation and management of adults and children presenting with a thrombocytosis.

Guy’s and St Thomas’ NHS Foundation Trust, London, Russells Hall Hospital, Dudley, West Midlands, Arrowe Park Hospital Arrowe Park Road Upton Wirral, Wellcome Trust Sanger Institute, Hinxton, Cambridge, St. James Hospital, James Street, Dublin, Gartnavel General Hospital 21 Shelley Road Glasgow, Addenbrooke’s Hospital, Cambridge, Salisbury Healthcare NHS Trust, Salisbury, Wiltshire, Cambridge Institute for Medical Research, Hills Road, Cambridge, John Radcliffe Hospital, Headley Way, Headington, Oxford, Royal Infirmary, Little France Crescent, Edinburgh, Sandwell and West Birmingham Hospitals, Dudley Road, Birmingham, Royal Hallamshire Hospital, Glossop Road, Sheffield, and Belfast City Hospital, Lisburn Road Belfast, UK

Resection Alone in 58 Children With Limited Stage, Lymphocyte-predominant Hodgkin Lymphoma-Experience From the European Network Group on Pediatric Hodgkin Lymphoma

Lymphocyte‐predominant Hodgkin lymphoma (LPHL) is a rare, CD20‐positive, good prognostic lymphoma in children. Patients with early‐stage LPHL who underwent successful surgical lymph node resection alone have been reported. To clarify the optimum treatment strategy in children, European study groups were asked to report their experience of surgery alone used in the treatment of pediatric LPHL.

Guideline for the diagnosis and management of myelofibrosis

The guideline group regarding the diagnosis and management of myelofibrosis was selected to be representative of UK‐based medical experts, together with a contribution from a single expert from the USA. MEDLINE and EMBASE were searched systematically for publications in English from 1966 until August 2011 using a variety of key words. The writing group produced the draft guideline, which was subsequently revised by consensus of the members of the General Haematology and Haemato‐oncology Task Forces of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The criteria used to state levels and grades of evidence are as outlined in the Procedure for Guidelines commissioned by the BCSH; the ‘GRADE‘ system was used to score strength and quality of evidence. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of primary myelofibrosis, as well as post‐polycythaemic myelofibrosis (post‐PV MF) and post‐thrombocythemic myelofibrosis (post‐ET MF) in both adult and paediatric patients.

A base substitution (T→C) in codon 29 of the α2-globin gene causes α thalassaemia

Summary. We have identified three individuals of Greek or Greek Cypriot origin with an atypical form of HbH disease characterized by a severe hypochromic microcytic anaemia associated with relatively small amounts of HbH in the peripheral blood. Molecular analysis has shown that each is a compound heterozygote for a previously described mutation affecting the poly A addition signal (AATAAA→ÁTAAG) and a previously undescribed mutation involving a T→C transition in codon 29 of the α2 gene causing a leucine→pro‐line substitution. Although this mutation would be expected to produce an unstable haemoglobin and hence a haemolytic anaemia, simple heterozygotes for the α29Leu→Pro mutation have the phenotype of α‐thalassaemia trait.

Outcome of children with nodular lymphocyte predominant Hodgkin lymphoma – a Children's Cancer and Leukaemia Group report

This report describes the clinical outcomes and follow‐up records of 42 children with nodular lymphocyte predominant Hodgkin lymphoma (LPHL) treated on United Kingdom Childrens Cancer Study Group (UKCCSG) HD1 (1982–1992) and HD2 protocols (1992–2000). The clinical records of 42 children with LPHL treated between 1982 and 2000 were reviewed retrospectively. All 42 had histology reviewed centrally and confirmed as LPHL by an expert panel. In both trials, only patients with stage IA disease had the option of being treated with either involved field radiation alone or combination chemotherapy consisting of chlorambucil, vinblastine, procarbazine and prednisolone (ChlVPP). Patients with all other stages were treated with ChlVPP chemotherapy. Thirty‐five patients (83%) presented with early stage disease (Stages I & II). All 42 patients achieved a complete remission (CR). Six children relapsed after primary therapy. The 5‐ and 10‐year relapse‐free survival rates were 87% and 82% respectively. Forty‐one are currently alive in CR. In conclusion, children with low‐stage LPHL treated between 1982 and 2000 according to the UK strategy for classical Hodgkin lymphoma (HL) had an excellent prognosis. There have been no second malignancies or transformations to B‐cell non‐Hodgkin lymphoma.

Beta thalassaemia in the indigenous British population.

Summary. We have analysed the molecular basis of β‐thalassaemia in 22 Anglo‐Saxon individuals, all of whom were heterozygous for β‐thalassaemia except for one, who was a compound heterozygote. Using a combination of allele‐specific priming of the polymerase chain reaction (PCR) and direct sequencing of genomic DNA amplied by the PCR, 20/ 23 β‐thalassaemic genes were characterized. Nine different mutations were identified; four are commonly found in the Mediterranean, one in Asia, one has been described previously in both Europe and Asia, and three are rare mutations associated with a dominant β‐thalassaemia phenotype. In three individuals the mutation remains uncharacterized despite sequence analysis of the β2‐globin gene and its immediate flanking regions. We report our findings and discuss the diversity of these mutations.

A NOVEL MUTATION (NONSENSE β 127) IN EXON 3 OF THE β GLOBIN GENE PRODUCES A VARIABLE THALASSAEMIC PHENOTYPE

Auerbach. A.D.. Rogatko. A. & Schroeder-Kurth. T.M. (1989) International Fanconi anemia registry: relation of clinical symptoms to diepoxybutane sensitivity. Blood. 73, 39 1-396. Hennett. J.M.. Catovsky. D.. Daniel, M.T. & Flandrin. G. (1982) Proposals for the classification of the myelodysplastic syndromes. British journalo/ Hacmatology. 51, 1 89199. dc Planque. M.M.. Kluin-Nelemans. H.C.. van Kriekan, H.J.M.. Kluin. P.M.. Brand. A . . Beverstock. G.C.. Willemze. R. & van Rood, J.J. ( 1988) Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults. British journal of

A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.

Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

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.

Incidence, management, and outcome of high-grade transformation of nodular lymphocyte predominant Hodgkin lymphoma: long-term outcomes from a 30-year experience.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare form of Hodgkin lymphoma that typically presents as early stage, indolent disease in young adult males. The relationship between NLPHL and DLBCL is incompletely understood, and there remains a paucity of data with regard the incidence and management of high‐grade transformation. We report the largest study to date describing the incidence, management and long‐term outcome of 26 cases of high‐grade transformation of NLPHL over a 30‐year period. We report a transformation incidence of 17.0%. Bone marrow, splenic, and liver infiltration with DLBCL was frequent. Patients with an aa‐IPI 2–3 have poorer OS and PFS (P = 0.034 and P = 0.009, respectively). Although the approach to treatment was somewhat variable, typically young, otherwise fit patients received anthracycline‐based induction, platinum‐based consolidation with stem cell harvesting, followed by autologous SCT with BEAM conditioning. Long‐term (5 year) PFS was over 60% with this approach, and comparable to our de novo DLBCL historical age and time period‐matched cohort largely treated with CHOP‐like chemotherapy alone. The transformation rate of 17.0% highlights the importance of accurate initial diagnosis, long‐term follow‐up, and re‐biopsy at relapse. Am. J. Hematol. 90:E103–E110, 2015.