Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial aplasia and myelodysplasia.

Aplastic anemia (AA) and myelodysplasia (MDS) are forms of bone marrow failure that are often part of the same progressive underlying disorder. While most cases are simplex and idiopathic, some show a clear pattern of inheritance; therefore, elucidating the underlying genetic cause could lead to a greater understanding of this spectrum of disorders. We used a combination of exome sequencing and SNP haplotype analysis to identify causative mutations in a family with a history of autosomal-dominant AA/MDS. We identified a heterozygous mutation in SRP72, a component of the signal recognition particle (SRP) that is responsible for the translocation of nascent membrane-bound and excreted proteins to the endoplasmic reticulum. A subsequent screen revealed another autosomal-dominant family with an inherited heterozygous SRP72 mutation. Transfection of these sequences into mammalian cells suggested that these proteins localize incorrectly within the cell. Furthermore, coimmunoprecipitation of epitope-tagged SRP72 indicated that the essential RNA component of the SRP did not fully associate with one of the SRP72 variants. These results suggest that inherited mutations in a component of the SRP have a role in the pathophysiology of AA/MDS, identifying a third pathway for developing these disorders alongside transcription factor and telomerase mutations.

Similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. A study on behalf of the UK Paediatric BMT Working Party, Paediatric Diseases Working Party and Severe Aplastic Anaemia Working Party of EBMT.

We explored the feasibility of unrelated donor haematopoietic stem cell transplant (HSCT) upfront without prior immunosuppressive therapy (IST) in paediatric idiopathic severe aplastic anaemia (SAA). This cohort was then compared to matched historical controls who had undergone first-line therapy with a matched sibling/family donor (MSD) HSCT (n = 87) or IST with horse antithymocyte globulin and ciclosporin (n = 58) or second-line therapy with unrelated donor HSCT post-failed IST (n = 24). The 2-year overall survival in the upfront cohort was 96 ± 4% compared to 91 ± 3% in the MSD controls (P = 0·30) and 94 ± 3% in the IST controls (P = 0·68) and 74 ± 9% in the unrelated donor HSCT post-IST failure controls (P = 0·02).The 2-year event-free survival in the upfront cohort was 92 ± 5% compared to 87 ± 4% in MSD controls (P = 0·37), 40 ± 7% in IST controls (P = 0·0001) and 74 ± 9% in the unrelated donor HSCT post-IST failure controls (n = 24) (P = 0·02). Outcomes for upfront-unrelated donor HSCT in paediatric idiopathic SAA were similar to MSD HSCT and superior to IST and unrelated donor HSCT post-IST failure. Front-line therapy with matched unrelated donor HSCT is a novel treatment approach and could be considered as first-line therapy in selected paediatric patients who lack a MSD.

NUP98-NSD1 fusion in association with FLT3-ITD mutation identifies a prognostically relevant subgroup of pediatric acute myeloid leukemia patients suitable for monitoring by real time quantitative PCR.

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3-ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98-NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98-NSD1 positive patients post-treatment.

Routine management, healthcare resource use and patient and carer-reported outcomes of patients with transfusion-dependent ß-thalassaemia in the United Kingdom: A mixed methods observational study.

Objectives: We evaluated routine healthcare management, clinical status and patient- and carer-reported outcomes in UK paediatric and adult patients with transfusion-dependent ß-thalassaemia (TDT). Methods: A multi-centre, observational mixed-methodology study evaluated 165 patients (50% male; median age 24.1 [interquartile range (IQR)] 11.8-37.2] years) from nine UK centres. Results: Patients had a mean of 13.7 (standard deviation [SD] ±3.2) transfusion episodes/year (mean retrospective observation period 4.7 [±0.7] years). The median (IQR) for iron overload parameters at the last assessment during the observation period were: serum ferritin (n = 165) 1961.0 (1090.0-3003.0) µg/L (38% > 2500 µg/L); R2 liver iron (n = 119) 5.4 (2.9-11.6) mg/g (16% ≥15 mg/g); T2* cardiac iron (n = 132) 30.3 (22.0-37.1) ms (10% < 10 ms). All patients received ≥1 iron chelator during the observation period; 21% received combination therapy. Patients had a mean of 7.8 (±8.1) non-transfusion-related hospital attendances or admissions/year. Adult patients' mean EQ-5D utility score was 0.69 (±0.33; n = 94 [≥16 years]) and mean Transfusion-dependent quality of life score was 58.6 (±18.4; n = 94 [≥18 years]). For Work Productivity and Activity impairment, mean activity impairment for patients ≥18 years (n = 88) was 48% (±32%) and for carers (n = 29) was 28% (±23%). Conclusions: TDT presents significant burden on patients, carers and healthcare resources.

British OsteoNEcrosis Study (BONES) protocol: a prospective cohort study to examine the natural history of osteonecrosis in older children, teenagers and young adults with acute lymphoblastic leukaemia and lymphoblastic lymphoma.

INTRODUCTION: Osteonecrosis is a well-recognised treatment-related morbidity risk in patients diagnosed with acute lymphoblastic leukaemia (ALL) and lymphoblastic lymphoma (LBL), with a high rate of affected patients requiring surgical intervention. Patients may have asymptomatic changes on imaging studies that spontaneously regress, and little is known about the natural history of osteonecrotic changes seen. The main aim of the British OsteoNEcrosis Study (BONES) is to determine the incidence of symptomatic and asymptomatic osteonecrosis in the lower extremities of survivors of ALL or LBL diagnosed aged 10-24 years in the UK at different time points in their treatment. This study also aims to identify risk factors for progression and the development of symptomatic osteonecrosis in this population, as well as specific radiological features that predict for progression or regression in those with asymptomatic osteonecrosis METHODS AND ANALYSIS: BONES is a prospective, longitudinal cohort study based at principal treatment centres around the UK. Participants are patients aged 10-24 years diagnosed with ALL or LBL under standard criteria. Assessment for osteonecrosis will be within 4 weeks of diagnosis, at the end of delayed intensification and 1, 2 and 3 years after the start of maintenance therapy. Assessment will consist of MRI scans of the lower limbs and physiotherapy assessment. Clinical and biochemical data will be collected at each of the time points. Bone mineral density data and vertebral fracture assessment using dual-energy X-ray absorptiometry will be collected at diagnosis and annually for 3 years after diagnosis of malignancy. ETHICS AND DISSEMINATION: Ethical approval has been obtained through the Yorkshire and Humber Sheffield Research Ethics Committee (reference number: 16/YH/0206). Study results will be published on the study website, in peer-reviewed journals and presented at relevant conferences and via social media. TRIAL REGISTRATION NUMBER: NCT02598401; Pre-results.

Detecting mismatch repair defects in myeloma.

Defects in the mismatch repair system are associated with a microsatellite unstable phenotype. In this chapter, we describe the preparation of purified plasma cells using CD138 magnetic microbeads as a source of tumor DNA. We also describe a robust, sensitive method for comparing microsatellite repeat units of tumor to constitutive DNA using polymerase chain reaction and laser scanning of fluorescently labeled amplicons in an automated sequencer in order to assess microsatellite instability in myeloma.

Introduction of sodium pentosan polysulfate and avoidance of urethral catheterisation: improved outcomes in children with haemorrhagic cystitis post stem cell transplant/chemotherapy.

PURPOSE: Haemorrhagic cystitis (HC) is an uncommon but potentially devastating complication of chemotherapy and bone marrow transplantation in children. We aimed to test the hypothesis that early recognition, sodium pentosan polysulfate (SPP), and avoidance of urethral catheterisation improve outcomes in children with HC. METHODS: A retrospective case note review was performed of all patients treated for HC in our hospital from 2002 to 2010. A protocol for the management of HC was introduced in 2007 advocating early detection, use of SPP, and avoidance of urethral catheterisation. Data collected on each patient included primary condition, medications at onset, blood transfusions, duration of symptoms, catheter usage, and outcome. Statistical analysis was performed using the Mann-Whitney U test, and Fisher's Exact test as appropriate, P < .05 being significant. RESULTS: Five patients were treated using protocol with 5 historical controls. There was no significant difference between the ages of the group, diagnosis, and treatment at onset of HC. In the historical group, 4 of 5 died with HC, but all recovered in the protocol group (P < .05). Blood transfusion requirements were also significantly reduced after protocol introduction (P < .05). CONCLUSION: Early identification, avoidance of urethral catheterisation, and use of SPP significantly reduces blood transfusion requirements and mortality from HC.

Two cases of aspergillus endocarditis in non neutropenic children on chemotherapy for acute lymphoblastic leukaemia.

Fungal endocarditis (FE) is a rare complication in immunocompromised patients which is difficult to diagnose and has been characterized by excessive mortality (> 50%) and morbidity, regardless of treatment. The lack of clinical trials due to the small number of cases contributes further to a poor outcome. In our two cases of aspergilllus endocarditis we reviewed the clinical features, echocardiographic findings, microbiologic data, treatment, and outcome of these 2 cases and provide a current characterization of the syndrome. In this paper we have demonstrated the diversity of presentation of a critical fungal infection in immunocompromised but non neutropenic paediatric patients. The prompt diagnosis and initiation of treatment is crucial for a favourable outcome along with the use of double antifungal treatment with liposomal amphotericin and voriconazole initially which could be later switched to oral voriconazole with a good tissue penetration. Histological samples as well as radiological evidence and echocardiograms should be reviewed by experienced clinicians in order to aid diagnosis and promptly initiate treatment for these patients in order to achieve a favourable outcome.

Less than half of patients aged 65 years or under with myeloma proceed to transplantation: results of a two region population-based survey.

In this population-based survey covering two geographically distinct UK regions, we evaluated the number of myeloma patients aged < or =65 years who have not undergone transplantation. The combined data from both of these regions showed that 57% of age-eligible patients were not transplanted. While early death and comorbidity accounted for nearly half of the non-transplanted patients, we examined the other reasons for non-transplantation within each region, assessed regional variations in reasons for non-transplant and looked at possible strategies aimed at increasing the transplantation rate.

DNA mismatch repair pathway defects in the pathogenesis and evolution of myeloma.

Genetic instability is a prominent feature in multiple myeloma and progression of this disease from monoclonal gammopathy of uncertain significance (MGUS) and smouldering myeloma (SMM) is associated with increasing molecular and chromosomal abnormalities. The DNA mismatch repair (MMR) pathway is a post-replicational DNA repair system that maintains genetic stability by repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication. Deficiencies in proteins pivotal to this pathway result in a higher mutation rate, particularly at regions of microsatellite DNA. We have investigated the proficiency of the MMR pathway in clinical samples and myeloma cell lines. Microsatellite analysis showed instability at one or more of nine loci examined in 15 from 92 patients: 7.7% of MGUS/SMM, 20.7% of MM/plasma cell leukaemia (PCL) and 12.5% of relapsed MM/PCL. An in vitro heteroduplex G/T repair assay found reduced repair in two cell lines, JIM1 and JIM3, and in two of four PCL cases and was associated with aberrant expression of at least one mismatch repair protein. Thus we show that MMR defects are found in plasma cell dyscrasias and the increased frequency during more active stages of the disease suggests a contributory role in disease progression.