Guidelines for the diagnosis and management of adult aplastic anaemia: A British Society for Haematology Guideline.

Pancytopenia with hypocellular bone marrow is the hallmark of aplastic anaemia (AA) and the diagnosis is confirmed after careful evaluation, following exclusion of alternate diagnosis including hypoplastic myelodysplastic syndromes. Emerging use of molecular cyto-genomics is helpful in delineating immune mediated AA from inherited bone marrow failures (IBMF). Camitta criteria is used to assess disease severity, which along with age and availability of human leucocyte antigen compatible donor are determinants for therapeutic decisions. Supportive care with blood and platelet transfusion support, along with anti-microbial prophylaxis and prompt management of opportunistic infections remain key throughout the disease course. The standard first-line treatment for newly diagnosed acquired severe/very severe AA patients is horse anti-thymocyte globulin and ciclosporin-based immunosuppressive therapy (IST) with eltrombopag or allogeneic haemopoietic stem cell transplant (HSCT) from a matched sibling donor. Unrelated donor HSCT in adults should be considered after lack of response to IST, and up front for young adults with severe infections and a readily available matched unrelated donor. Management of IBMF, AA in pregnancy and in elderly require special attention. In view of the rarity of AA and complexity of management, appropriate discussion in multidisciplinary meetings and involvement of expert centres is strongly recommended to improve patient outcomes.

Role of allogeneic transplantation in chronic myelomonocytic leukemia: an international collaborative analysis.

To determine the survival benefit of allogeneic hematopoietic cell transplantation (allo-HCT) in chronic myelomonocytic leukemias (CMML), we assembled a retrospective cohort of CMML patients 18-70 years old diagnosed between 2000 and 2014 from an international CMML dataset (n = 730) and the EBMT registry (n = 384). The prognostic impact of allo-HCT was analyzed through univariable and multivariable time-dependent models and with a multistate model, accounting for age, sex, CMML prognostic scoring system (low or intermediate-1 grouped as lower-risk, intermediate-2 or high as higher-risk) at diagnosis, and AML transformation. In univariable analysis, lower-risk CMMLs had a 5-year overall survival (OS) of 20% with allo-HCT vs 42% without allo-HCT (P < .001). In higher-risk patients, 5-year OS was 27% with allo-HCT vs 15% without allo-HCT (P = .13). With multistate models, performing allo-HCT before AML transformation reduced OS in patients with lower-risk CMML, and a survival benefit was predicted for men with higher-risk CMML. In a multivariable analysis of lower-risk patients, performing allo-HCT before transformation to AML significantly increased the risk of death within 2 years of transplantation (hazard ratio [HR], 3.19; P < .001), with no significant change in long-term survival beyond this time point (HR, 0.98; P = .92). In higher-risk patients, allo-HCT significantly increased the risk of death in the first 2 years after transplant (HR 1.46; P = .01) but not beyond (HR, 0.60; P = .09). Performing allo-HCT before AML transformation decreases life expectancy in lower-risk patients but may be considered in higher-risk patients.

Red cell transfusion in outpatients with myelodysplastic syndromes: a feasibility and exploratory randomised trial.

Optimal red cell transfusion support in myelodysplastic syndromes (MDS) has not been tested and established. The aim of this study was to demonstrate feasibility of recruitment and follow-up in an outpatient setting with an exploratory assessment of quality of life (QoL) outcomes (EORTC QLQ-C30 and EQ-5D-5L). We randomised MDS patients to standardised transfusion algorithms comparing current restrictive transfusion thresholds (80 g/l, to maintain haemoglobin 85-100 g/l) with liberal thresholds (105 g/l, maintaining 110-125 g/l). The primary outcomes were measures of compliance to transfusion thresholds. Altogether 38 patients were randomised (n = 20 restrictive; n = 18 liberal) from 12 participating sites in UK, Australia and New Zealand. The compliance proportion for the intention-to-treat population was 86% (95% confidence interval 75-94%) and 99% (95-100%) for restrictive and liberal arms respectively. Mean pre-transfusion haemoglobin concentrations for restrictive and liberal arms were 80 g/l (SD6) and 97 g/l (SD7). The total number of red cell units transfused on study was 82 in the restrictive and 192 in the liberal arm. In an exploratory analysis, the five main QoL domains were improved for participants in the liberal compared to restrictive arm. Our findings support the feasibility and need for a definitive trial to evaluate the effect of different red cell transfusion thresholds on patient-centred outcomes.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Iron chelation therapy in low risk myelodysplastic syndrome.

Anaemia is the commonest cytopenia seen in patients with myelodysplastic syndrome (MDS), and the majority of patients will require transfusion support at some point. Blood transfusions are rich in iron, which leads to the accumulation of body iron over time. It is accepted that this ultimately causes end organ damage and may impact on both morbidity and mortality. In addition, recent data has increased our interest in the subject with regard to the potential impact on stem cell transplant outcome and an anti-leukaemic effect of iron chelation therapy. There is still debate over which patients should receive iron chelation therapy, but the emergence of new diagnostic and prognostic markers in MDS may help decision making in the clinic setting.

CSNK1A1 mutations and gene expression analysis in myelodysplastic syndromes with del(5q).

Mutations of CSNK1A1, a gene mapping to the commonly deleted region of the 5q- syndrome, have been recently described in patients with del(5q) myelodysplastic syndromes (MDS). Haploinsufficiency of Csnk1a1 in mice has been shown to result in ß-catenin activation and expansion of haematopoietic stem cells (HSC). We have screened a large cohort of 104 del(5q) MDS patients and have identified mutations of CSNK1A1 in five cases (approximately 5%). We have shown up-regulation of ß-catenin target genes in the HSC of patients with del(5q) MDS. Our data further support a central role of CSNK1A1 in the pathogenesis of MDS with del(5q).

Prospective study of rabbit antithymocyte globulin and cyclosporine for aplastic anemia from the EBMT Severe Aplastic Anaemia Working Party.

Rabbit antithymocyte globulin (rATG; thymoglobulin, Genzyme) in combination with cyclosporine, as first-line immunosuppressive therapy, was evaluated prospectively in a multicenter, European, phase 2 pilot study, in 35 patients with aplastic anemia. Results were compared with 105 age- and disease severity-matched patients from the European Blood and Marrow Transplant registry, treated with horse ATG (hATG; lymphoglobulin) and cyclosporine. The primary end point was response at 6 months. At 3 months, no patients had achieved a complete response to rATG. Partial response occurred in 11 (34%). At 6 months, complete response rate was 3% and partial response rate 37%. There were 10 deaths after rATG (28.5%) and 1 after subsequent HSCT. Infections were the main cause of death in 9 of 10 patients. The best response rate was 60% for rATG and 67% for hATG. For rATG, overall survival at 2 years was 68%, compared with 86% for hATG (P = .009). Transplant-free survival was 52% for rATG and 76% for hATG (P = .002). On multivariate analysis, rATG (hazard ratio = 3.9, P = .003) and age more than 37 years (hazard ratio = 4.7, P = .0008) were independent adverse risk factors for survival. This study was registered at www.clinicaltrials.gov as NCT00471848.

Targeted re-sequencing analysis of 25 genes commonly mutated in myeloid disorders in del(5q) myelodysplastic syndromes.

Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at great depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 cases of early or advanced del(5q) myelodysplastic syndromes. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases had at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in cases of 5q- syndrome (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. Fifty-two percent of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations had allele frequencies <20%, likely below the detection limit of traditional sequencing methods. Genomic array data showed that cases of advanced del(5q) myelodysplastic syndrome had a complex background of cytogenetic aberrations, often encompassing genes involved in myeloid disorders. Our study is the first to investigate the molecular pathogenesis of early and advanced del(5q) myelodysplastic syndromes using next-generation sequencing technology on a large panel of genes frequently mutated in myeloid malignancies, further illuminating the molecular landscape of del(5q) myelodysplastic syndromes.

Convergent somatic evolution commences in utero in a germline ribosomopathy.

Clonal tracking of cells using somatic mutations permits exploration of clonal dynamics in human disease. Here, we perform whole genome sequencing of 323 haematopoietic colonies from 10 individuals with the inherited ribosomopathy Shwachman-Diamond syndrome to reconstruct haematopoietic phylogenies. In ~30% of colonies, we identify mutually exclusive mutations in TP53, EIF6, RPL5, RPL22, PRPF8, plus chromosome 7 and 15 aberrations that increase SBDS and EFL1 gene dosage, respectively. Target gene mutations commence in utero, resulting in a profusion of clonal expansions, with only a few haematopoietic stem cell lineages (mean 8, range 1-24) contributing ~50% of haematopoietic colonies across 8 individuals (range 4-100% clonality) by young adulthood. Rapid clonal expansion during disease transformation is associated with biallelic TP53 mutations and increased mutation burden. Our study highlights how convergent somatic mutation of the p53-dependent nucleolar surveillance pathway offsets the deleterious effects of germline ribosomopathy but increases opportunity for TP53-mutated cancer evolution.

Cardiac iron overload in transfusion-dependent patients with myelodysplastic syndromes.

Transfusion-dependent myelodysplastic (MDS) patients are prone to iron overload. We evaluated 43 transfused MDS patients with T2* magnetic resonance imaging scans. 81% had liver and 16·8% cardiac iron overload. Liver R2* (1000/T2*), but not cardiac R2*, was correlated with number of units transfused (r=0·72, P<0·0001) and ferritin (r=0·53, P<0·0001). The area under the curve of a time-ferritin plot was found to be much greater in patients with cardiac iron loading (median 53·7x10(5) Megaunits vs. 12·2x10(5) Megaunits, P=0·002). HFE, HFE2, HAMP or SLC40A1 genotypes were not predictors of iron overload in these patients.

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.

Marked downregulation of the granulopoiesis regulator LEF1 is associated with disease progression in the myelodysplastic syndromes.

Lymphoid enhancer-binding factor 1 (LEF1) is a neutrophilic granulopoiesis regulator whose absence is critical in congenital neutropenia. We have shown LEF1 downregulation in the CD34(+) cells of the majority of myelodysplastic syndromes (MDS) patients. LEF1 was the most significant differentially expressed gene between early and advanced MDS. Marked LEF1 downregulation was found in 27/32 patients with advanced MDS and in 6/35 patients with early MDS, and was associated with neutropenia. Downregulation of LEF1 mRNA was reflected at the protein level. Immunostaining for CD34/LEF1 may represent a marker of advanced MDS. LEF1 may play a role in the defective maturation of myeloid progenitors in MDS.

Valuation of transfusion-free living in MDS: results of health utility interviews with patients.

BACKGROUND: This study measured how myelodysplastic syndrome (MDS) patients value transfusion independence (TI), reduced transfusions (RT) and transfusion-dependence (TD) using health utility assessment methodology. METHODS: 47 MDS patients were interviewed, US (n = 8), France (n = 9), Germany (n = 9) and the UK (n = 21), to elicit the utility value of TI, RT and TD. Health states were developed based on literature; patient forum discussions; and were validated by a hematologist. Face-to-face interviews used the feeling thermometer Visual Analogue Scale (VAS) and the Time Trade-Off (TTO) method to value the health states on a 0 (dead) to 1 (perfect health) scale. Socio-demographic, clinical, and quality-of-life (EQ-5D) characteristics were surveyed to describe the patient sample. RESULTS AND DISCUSSION: The mean age was 67 years (range: 29-83); 45% male, 70% retired; 40% had secondary/high school education, or higher (32%), and 79% lived with family, a partner or spouse, or friends. The mean time from MDS diagnosis was 5 years (range:1-23). Most patients (87%) received previous transfusions and 49% had received a transfusion in the last 3 months. Mean EQ-5D index score was 0.78; patients reported at least some problem with mobility (45%), usual activities (40%), pain/discomfort (47%), and anxiety/depression (34%). Few patients had difficulty understanding the VAS (n = 3) and TTO (n = 4) exercises. Utility scores for TI were higher than for RT (0.84 vs. 0.77; p < 0.001) or TD (0.84 vs. 0.60; p < 0.001). Three patients rated TD worse than dead. Corresponding VAS scale scores were 78 vs. 56; (p < 0.001), and 78 vs. 31 (p < 0.001), respectively. CONCLUSION: Patients value TI, suggesting an important role for new treatments aiming to achieve greater TI in MDS. These results can be used in preference-based health economic evaluation of new MDS treatments, such as in future cost-utility studies.