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.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders.

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.

This document represents an update of the British Society of Haematology guideline published in 2014 due to advances in understanding the biology and therapy of the myelodysplastic syndromes (MDS).1 The objective of these guidelines is to provide healthcare professionals with clear guidance on the diagnosis and evaluation of prognosis of adult patients with MDS. A separate BSH guideline covers the Management of Adult MDS which is published alongside this guideline. A separate good practice paper detailing the management of patients with chronic myelomonocytic leukaemia (CMML) will follow and is not considered in these guidelines. These guidelines were compiled according to the BSH process https://b-s-h.org.uk/media/16732/bsh-guidance-development-process-dec-5-18.pdf. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. The guideline group was selected to be representative of UK medical experts and the manuscript was reviewed by the UK MDS Patient Support Group. Recommendations are based on a review of the literature using Medline/Pubmed searches. Search terms included: Myelodysplasia, MDS, myelodysplastic, refractory an(a)emia, refractory cytopenia, deletion 5q, del(5q), idiopathic cytopenia of undetermined significance (ICUS), clonal cytopenia of undetermined significance (CCUS), clonal haematopoiesis of indeterminate potential (CHIP), diagnosis, diagnostic, investigation, cytogenetic, molecular, mutation, bone marrow, flow cytometry risk, prognosis. Only English-language publications from January 2012 to December 2020 were included in the literature search. Additional searches and subsection heading terms were conducted by members of the writing committee at the time of final submission to the British Journal of Haematology. Titles and/or abstracts of publications obtained from the database searches described were curated and manually reviewed by members of the writing committee. Review of the manuscript was performed by the BSH Guidelines Committee Haemato-oncology Task Force, the BSH Guidelines Committee and the haemato-oncology sounding board of the BSH. It was also posted on the members section of the BSH website for comment. This guideline has also been reviewed by patient representatives from the MDS UK Patient Support Group (mdspatientsupport.org.uk). These organisations do not necessarily endorse the contents. The myelodysplastic syndromes (MDS) are a group of clonal bone marrow neoplasms characterised by ineffective haematopoiesis and manifested by morphological dysplasia in haematopoietic cells and by peripheral cytopenia(s).2 They have a variable predilection for the development of acute myeloid leukaemia (AML). The incidence of MDS in the UK is 3·72/100,000 population/year; it is predominantly a disease of the elderly (median age at diagnosis 75·7 years) and more common in men (approximately 2:1).3 Patients with suspected MDS should be assessed by a haematologist with a specialist interest in the disease. They should be referred for a second opinion to a regional or national centre when required by the clinician, or requested by the patient. All patients with a diagnosis of MDS must be discussed at a multidisciplinary team meeting (MDT), which should include allogeneic stem cell transplantation representation. All patients diagnosed with MDS should be reported to the National Cancer Registry, via the MDT, and to MDS-specific registries if appropriate. Myelodysplastic syndrome is defined by a combination of cytopenias and morphological bone marrow dysplasia. Myelodysplastic syndromes should be considered in all patients with otherwise unexplained cytopenia(s). World Health Organisation (WHO) thresholds for cytopenias are haemoglobin <100 g/l, absolute neutrophil count <1·8 × 109/l and platelets <100 × 109/l.2 However, higher values (as defined by local laboratory ranges) do not exclude the diagnosis if definitive morphological and/or cytogenetic abnormalities are present. A diagnostic algorithm for suitable patients is outlined in Fig. 1. Table I shows the minimum clinical assessment and laboratory investigation of a patient with possible MDS. Selected patients may require further investigations (Table II). Alternative causes of marrow dysplasia should also be considered. In the context of persistent and otherwise unexplained cytopenias, a WHO-defined diagnosis of MDS requires either (i) morphological dysplasia (involving ≥10% of bone marrow cells in ≥1 lineage); (ii) increased myeloblasts (≥5%, but <20%); or (iii) evidence of clonality with a typical MDS-associated cytogenetic abnormality.2, 4 Dysplasia is not restricted to MDS patients and can occur following a toxic insult, in reactive conditions or secondary to haematinic deficiencies. Furthermore, dysplasia has been reported in healthy individuals.5, 6 Identifying MDS can therefore be challenging and caution is required when the diagnosis is based solely on morphology, particularly in borderline cases or those with unilineage dysplasia. Other causes of morphological dysplasia should be excluded and a period of observation followed by repeat sampling may be warranted. New technologies, in particular genomic testing, may help in challenging cases by providing additional markers of clonality. Although the presence of clonal markers should not be considered in isolation of other diagnostic modalities, there are strong associations between particular genetic lesions (for example mutations in SF3B1 or isolated deletion of chromosome 5q) with WHO-defined MDS subtypes. In patients with <10% marrow dysplasia and lacking a clonal abnormality, the term ‘idiopathic cytopenia of undetermined significance’ (ICUS) may be used where cytopenias are sustained (>6 months) and there is no other identifiable cause.7 Such patients should be observed (with repeat investigation if necessary) for subsequent development of overt MDS. Chronic myelomonocytic leukaemia (CMML) has been reclassified to the WHO subgroup of myelodysplastic/myeloproliferative neoplasms (MDS/MPN)2 and is not considered further in this guideline. In confirmed cases of MDS, family history and clinical features should be reviewed to identify those with germline predisposition, which may have implications for prognosis, genetic counselling and management. Both blood film and bone marrow examination by a haematologist or haematopathologist with experience in diagnosing MDS, looking for characteristic morphological features of dysplasia, are necessary for diagnosis, classification and prognostic evaluation of MDS. Blood films should be assessed for dysplasia in erythroid, platelet and white-cell lineages.2, 8 Bone marrow examination of May–Grünwald–Giemsa (or equivalent)-stained smears should routinely comment on myeloid, megakaryocyte and erythroid maturation, and report dysplasia if present. Blast percentage should be enumerated. Optimal differential count should evaluate 500 or more nucleated cells, including 30 or more megakaryocytes. Good quality smears and stains are essential for accurate diagnosis. Fresh specimens should be processed within 2 hours, where possible, and excess of ethylenediamine tetra-acetic acid (EDTA) should be strictly avoided. Stains should be well controlled and checked by examining non-MDS films. Prussian Blue or Perls’ stain should be performed on all marrow aspirates to assess iron stores and to quantitate ring sideroblasts. In the revised WHO classification,2 the presence of an SF3B1 mutation reduces the ring sideroblast percentage threshold required for a diagnosis of MDS with ring sideroblasts (MDS-RS) from 15% to 5%.2 A trephine biopsy (decalcified, paraffin or plastic-embedded) should be taken from all patients and sectioned for analysis alongside the aspirate. Whilst dysplasia can be harder to assess, the histology of the trephine section provides supportive information for diagnosis, including architectural disruption (e.g. disruption of erythroid islands; abnormal localisation of immature precursors), cellularity and fibrosis (with reticulin staining). Trephine section histology is especially helpful for the diagnosis of hypocellular MDS and MDS/myeloproliferative neoplasms (MPN) overlap syndromes.9 Patients with MDS/MPN overlap including CMML are now considered a distinct entity by the WHO when features of both MDS and MPN are present. This includes MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) which may evolve from MDS-RS. Around 10–20% of patients with MDS have decreased marrow cellularity.10 The WHO classification of myeloid neoplasm terms this hypoplastic MDS (h-MDS), although it does not give it a distinct category.2 Hypocellularity in MDS can present diagnostic difficulties with other bone marrow failure (BMF) syndromes especially aplastic anaemia. A study integrating cytohistological and genetic features in adult patients with hypocellular bone marrows has led to proposed criteria to define h-MDS.10 This separates patients into two distinct groups, one with features highly consistent with myeloid neoplasm and one more consistent with a non-malignant BMF. The two groups have significantly different risk of blast progression and overall survival (OS). Flow cytometry should be performed for paroxysmal nocturnal haemoglobinuria in patients with h-MDS. Enumeration of blast percentage should be undertaken by morphological assessment of the bone marrow aspirate. This is considered the gold standard. However, if the aspirate smear is suboptimal, then the bone marrow trephine section may be used to quantitate blasts using immunohistochemistry. There is no specific immunophenotypic finding diagnostic of MDS, and flow cytometry is therefore not mandatory. Aberrant flow cytometric profiles may support the diagnosis of MDS but should be interpreted with morphological and cytogenetic or molecular findings. Common findings are aberrant antigen expression on myeloid progenitors, maturing myeloid, monocytic and erythroid lineages, reduced numbers of B-cell progenitors,11 and increased CD34+ cells. Many cases also show lineage infidelity antigen expression. Flow cytometry can be useful to enumerate myeloid progenitor cells (CD34+ cells) which may in turn be a proxy for morphological blast percentage but these do not always correlate precisely, for example due to haemodilution of the aspirate or the progenitor cell phenotype lacking CD34 expression. Recommendations for standardisation of flow cytometric methodology, including consensus recommendations for cell sampling, handling and processing have been published;12-16 validation is ongoing. Chromosomal abnormalities evidencing a clonal disorder are detected by cytogenetic analyses in approximately 50% of MDS patients. Some recurrent abnormalities [most commonly, −5, del(5q), −7, del(7q), i(17q)] are considered MDS-defining in a cytopenic patient, even without morphological dysplasia (a comprehensive list is shown in Fig 1 and Table III).2, 17 G-banding or metaphase cytogenetic analysis should be performed on all suspected MDS cases to aid diagnosis, prognosis and inform management. When no abnormality is found in a diagnostic sample, a minimum of 20 metaphases should be examined and reported using International System for Human Cytogenetic Nomenclature Recommendations.18 Cytogenetic assessment is essential for international prognostic scoring systems.17 Furthermore, specific cytogenetic abnormalities may provide a marker for assessing response to therapy and evaluating residual disease. Since both the type and number of karyotypic abnormalities may have prognostic significance, adherence to International Working Group on MDS Cytogenetics consensus guidelines in the enumeration of abnormalities is recommended.19 <15% / <5%b b If SF3B1 mutation is present. BM <5%, PB <1%, No Auer rods <15% / <5%b b If SF3B1 mutation is present. BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods BM 5-9% or PB 2-4%, BM <10% and PB <5%, No Auer rods BM 10-19% or PB 5-19%, Or Auer rods BM and PB <20% BM <5%, PB <1% c c 1% PB blasts must be recorded on ≥2 separate occasions. , No Auer rods BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods In cases where G-banding analysis is not possible or fails, fluorescence in situ hybridisation (FISH) analysis of marrow aspirate or peripheral blood smears for selected common cytogenetic anomalies (e.g. −7, del(5q), +8) may be performed, to detect key abnormalities of prognostic significance or provide confirmation of clonality in borderline diagnostic cases. Where available, single nucleotide polymorphisms array analysis (SNP-A) can provide a more precise, genome-wide analysis which is independent of metaphases.20-22 Although not currently mandated in diagnostic work-up, this can provide useful additional information. In particular, where conventional cytogenetics fails SNP-A array can provide a full karyotype, and should be strongly considered in such cases. SNP-A may also detect karyotypic abnormalities in ~16–30% additional cases where they were not detected by metaphase cytogenetics (MC).20-22 Importantly, copy number abnormalities detected by SNP-A in cases where none were found by MC, are prognostic;23 thus prognostic equivalence can be reasonably assumed for larger structural abnormalities detected by this approach, and should be reported as such. This, however, cannot currently be assumed for smaller abnormalities below the detection resolution of conventional cytogenetics. SNP-A reports should state clearly those lesions considered detectable by MC and which should (and should not) be considered when calculating the cytogenetic risk score for current prognostic systems (e.g. Revised International Prognostic Scoring System [IPSS-R]). Furthermore, SNP-A have limited capacity for detecting translocations which are confined to those with associated microdeletions or uniparental disomy.24 Next-generation sequencing (NGS) has identified recurrent gene mutations in DNA from haematopoietic cells of ~90% of MDS patients, some of which may have independent prognostic significance.25-27 Molecular testing using targeted mutation panels is now widely available, increasingly affordable and should be considered in all patients (unless clearly not appropriate) for its potential to inform on diagnosis, prognosis and management. Sensitivity is highest on bone marrow, but can usefully be performed on peripheral blood in situations in which bone marrow biopsy is impractical or undesirable (provided that circulating myeloid cells are present). Patients should be counselled and at least verbal consent taken prior to genetic testing to explain the possible results including the implications of identifying a germline mutation. Detection of certain MDS-associated mutations can be used to establish subtypes with prognostic relevance. For example, SF3B1 mutations are found in >95% of MDS cases with ring sideroblasts, and are associated with a relatively favourable prognosis28 compared with SF3B1 wild-type MDS-RS cases.29 Due to its characteristic features SF3B1-mutated MDS has been proposed by The International Working Group as a distinct MDS subtype, although this is not yet formally incorporated into the WHO classification.30 TP53 mutations in MDS with isolated del(5q) helps identify early clonal evolution and predict disease progression and poorer prognosis in this generally favourable subgroup.31 In MDS more broadly, combinations of mutation, deletion and/or loss of heterozygosity events, resulting in ‘double-hit’ biallelic loss of TP53, are strongly associated with complex (typically monosomal) karyotype and exceptionally poor survival outcomes.32 In contrast, patients with single-hit, monoallelic TP53 mutations often lack associated chromosomal aneuploidies and display similar therapy response and outcomes to MDS patients without mutated TP53.32, 33 Mutations in genes such as ASXL1, EZH2 and RUNX1 confer adverse prognosis in univariate analysis but their prognostic significance in multivariate analysis has not yet been consistently reproduced in independent series.28, 34 Mutation status will likely inform prognosis in future models (e.g. IPSS-Molecular; in development) and guide eligibility for clinical trials of emerging targeted therapies (e.g. IDH1/IDH2 inhibitors; spliceosome inhibitors). In view of potential challenges of morphological diagnosis of MDS, mutation analysis can provide objective evidence of clonal disease. However, somatic mutations can be identified in healthy individuals and detection of mutations alone is not considered diagnostic.2 Notably, MDS patients tend to have a higher allele fraction and greater number of mutations than healthy, older individuals.35, 36 In an attempt to standardise testing, NHS England has created the NHS Genomic Medicine service, comprised of a national Genomic Laboratory Hub (GLH) network. A National Genomic Test Directory specifies genomic tests commissioned by the NHS in England and patients who are eligible for testing. Each GLH will provide cytogenetics and DNA sequencing with analysis and expert interpretation. Currently, those with suspected or confirmed MDS are eligible for a targeted NGS panel. Classification of MDS remains largely based upon morphological examination.2 The latest WHO revision has updated nomenclature and removed the focus on specific lineages of cytopenia (Table III and Fig 2).2 A WHO classification subtype should be recorded for every patient in the bone marrow report. In adult patients with at least 20% blasts the disease is classified as AML, although cases with 20–30% blasts were included in derivation of the IPSS. Myelodysplastic syndrome secondary to prior cytotoxic therapy is classified separately, under therapy-related myeloid neoplasms. Clonal haematopoiesis can be detected in the healthy population, typically with increasing age.37-40 This is frequently characterised by acquisition of MDS-associated mutations, but without other clinicopathological features of MDS. This has been termed ‘clonal haematopoiesis of indeterminate potential’ (CHIP) or ‘age-related clonal haematopoiesis’ (ARCH), and can be found in >10% of healthy individuals over 70 years of age.38 The most commonly identified mutations are in genes involved in epigenetic regulation (DNMT3A, TET2, ASXL1). These are commonly mutations in single genes only, at low allele frequency (<10%). Risk of transformation to haematological malignancy is low (<1% per year). Annual monitoring of blood counts in individuals found to have CHIP may, therefore, be appropriate. Factors that might increase risk of progression to myeloid malignancy include higher variant allele frequency, presence of multiple CHIP mutations or particular high-risk mutations (e.g. TP53, IDH2).35 A new nomenclature has emerged for conditions related to MDS but not fulfilling the formal diagnostic criteria (Table IV). These are increasingly used to describe observed states bearing isolated molecular, cytopenic or morphological features associated with MDS, and which might predispose to haematological malignancy. Clonal haematopoiesis of indeterminate potential Age-related clonal haematopoiesis ICUS carries approximately 9% risk of developing myeloid malignancy at 10 years.41 Evidence-based recommendations on monitoring cannot yet be made and decisions should be guided by the overall clinical picture and context; the possibility of non-MDS-related causes for the cytopenia should be reviewed during follow-up. In contrast, close monitoring of patients with CCUS is recommended, given emerging evidence that these patients carry a high — possibly universal — risk of progression to frank haematological malignancy.41 Beyond securing a diagnosis, identification of a germline condition underlying MDS can have important implications for treatment planning; for example, when selecting sibling donors for allogeneic stem cell transplantation. A three-generational family history should be taken. Table V outlines individuals in whom the possibility of a myeloid neoplasm with germline predisposition should be considered. Some germline mutations, such as those in TP53, RUNX1 and GATA2, may also be detected by NGS platforms aimed at detecting somatic mutations. Germline variants may be suggested by a variant allele frequency around 50%, although this can be the case too for dominant, deeply established somatic clones, so cannot alone be routinely taken as presumptive evidence. Early contact with a centre having clinical experience of constitutional marrow failure syndromes and a clinical genetics department is indicated in cases of suspected germline conditions. Patients and family members should ideally be offered genetic counselling before genetic screening if there is a high clinical suspicion.42 Since its publication in 1997, the IPSS has been an important tool for assessing the outcome of patients with untreated, primary adult MDS.43 Additional prognostic variables have been identified, the most important of which are newer cytogenetic groupings (Table VI) that give more accurate prognostic information.17 The IPSS-R described the relative importance of defined clinical factors to prognosis by multivariate analysis of 7012 primary, adult MDS patients not treated with disease-modifying therapies. Using the same parameters as the IPSS (cytogenetic groups, marrow blast percentage and cytopenias), it provided extended categorisation of cytogenetic subgroups, refinement of blast counts <5% and depth of cytopenias (Table VII).44 The IPSS-R stratifies into 5 risk categories and has improved the prognostic ability to determine survival and AML evolution in untreated adult patients with primary MDS (Table VIII). A web-based tool to calculate the IPSS-R can be accessed via the UK MDS Forum website (www.ukmdsforum.org.uk). In some head-to-head comparisons the IPSS-R has outperformed both the IPSS and WHO-based (WPSS) prognostic models, at least for some subgroups45-47 and is currently the recommended scoring system for determining prognosis. However, as long as NICE approval for azacitidine is based on IPSS risk, that earlier model retains clinical utility in the UK. Mutation data do not currently inform any prospectively validated prognostic scoring system in MDS. An IPSS-Molecular is currently under development. Consideration should be given to a regular review of prognosis for individual MDS patients. For example, loss of response to erythropoiesis stimulating agents or lenalidomide is associated with a reduction in overall survival. In contrast, dynamic IPSS or IPSS-R data indicate that for lower-risk MDS, the longer the patient remains low risk, the better the overall prognosis compared with the prognosis at diagnosis.48, 49 In lower-risk patients potentially eligible for allogeneic stem cell transplantation, consideration should be given to surveillance bone marrow testing. Although mathematical modelling of timing of transplantation was originally based on a move to transplant after AML transformation in lower-risk MDS, expert opinion would favour considering transplantation following identification of earlier signs of progression, such as increased bone marrow blast percentage, clonal evolution (cytogenetic/molecular), or increasing fibrosis in subtypes such as del(5q) MDS.50 Such surveillance should be in liaison with the transplant centre. All the authors contributed to the writing of these guidelines. The writing committee would like to thank: the team of MDS experts at the MDS UK Patient Support Group for their critical review of the manuscript on behalf of the MDS UK Patient Support Group, Jacky Wilson for her help in undertaking the initial literature review, also the BSH Haemato-oncology Task Force, the BSH sounding board and the BSH Guidelines Committee for their support in preparing this guideline. All authors and the MDS UK Patient Support Group have made a declaration of interests to the BSH and Task Force Chairs which may be viewed on request. Members of the writing group will inform the writing group Chair if any new evidence becomes available that would alter the strength of the recommendations made in this document or render it obsolete. The document will be reviewed regularly by the relevant Task Force and the literature search will be re-run every three years to search systematically for any new evidence that may have been missed. The document will be archived and removed from the BSH current guidelines website if it becomes obsolete. If new recommendations are made an addendum will be published on the BSH guidelines website.51 While the advice and information in this guidance is believed to be true and accurate at the time of going to press, neither the authors, the BSH nor the publishers accept any legal responsibility for the content of this guidance.

The myelodysplastic syndromes (MDS) are clonal disorders of haemopoiesis. They share characteristic morphological abnormalities of the blood and bone marrow and a risk of evolution to acute leukaemia, which varies depending on the MDS subtype. Most patients are elderly and present with symptoms of marrow failure despite increased marrow cellularity. The World Health Organization revised the classification of MDS in 2016, incorporating morphological, genetic and clinical features. The prognosis of the disease can be estimated using the Revised International Prognostic Scoring System, and appropriate treatment offered. This treatment should be tailored to the individual patient.

The myelodysplastic syndromes (MDS) are common myeloid malignancies characterized by ineffective hematopoiesis and blood cytopenias, with patients showing increasing bone marrow blasts with disease progression [1].Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1, and ZRSR2) are the most common mutations found in MDS, occurring in over 50% of all cases [2-4].There is evidence that some spliceosome components play a role in the maintenance of genomic stability [5].Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of Rloops (RNA-DNA hybrids with a displaced single stranded DNA) [6].Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loop formation in leukemia cell lines, resulting in increased DNA damage, replication stress, and activation of the ATR-Chk1 pathway [7,8].SF3B1 is the most frequently mutated

Red blood cell transfusions (RBCT) remain the cornerstone of supportive care in lower-risk myelodysplastic syndrome (LRMDS) [1]. Transfusion dependency in LRMDS patients is associated with inferior outcomes, mainly attributed to severe bone marrow failure [2]. However, iron toxicity, due to frequent RBCT or ineffective erythropoiesis, may be an additional negative prognostic factor [3,4,5,6]. Recently, much progress has been made in unraveling the iron metabolism. The peptide hormone hepcidin is the key regulator by inhibiting iron uptake through degradation of ferroportin, a cellular iron exporter [7]. Erythroferrone and GDF15, produced by erythroblasts, inhibit hepcidin production, which leads to increased uptake and cellular release of iron for the purpose of erythropoiesis [8]. The pathophysiology of iron metabolism in MDS is still not completely understood. Exceedingly high reactive oxygen species (ROS) levels are associated with iron toxicity, disease development, and progression in MDS patients [9,10,11,12]. Malondialdehyde (MDA), resulting from lipid peroxidation of polyunsaturated fatty acids, is a biomarker of oxidative stress [10, 12]. Currently, little is known about the prognostic impact of ROS in MDS patients. The aim of this study is twofold: (1) describe iron and oxidative stress parameters over time in LRMDS patients and (2) to assess their effect on overall and progression-free survival. The EUMDS registry prospectively collects observational data on newly diagnosed LRMDS patients from 148 centers in 16 countries in Europe and Israel as of January 2008. All patients provided informed consent. Clinical data were collected at baseline and at each six-monthly follow-up visit. Serum samples were collected prospectively at each visit from 256 patients included in six participating countries. Conventional iron parameters were measured with routine assays. We additionally analyzed hepcidin, growth differentiation factor 15 (GDF15), soluble transferrin receptor (sTfR), non-transferrin bound iron (NTBI), labile plasma iron (LPI), and MDA. Subjects were prospectively followed until death, loss to follow-up, or withdrawal of consent. All iron parameters were measured centrally at the department of Laboratory Medicine of the Radboudumc, Nijmegen, The Netherlands. Serum samples were collected just prior to transfusion in transfusion-dependent patients and stored at −80 °C. Details on the assays and reference ranges of hepcidin, GDF15, sTfR, NTBI, LPI, and MDA are provided in the supplement. The Spearman rank test was used to evaluate correlations between iron parameters. We stratified the results by transfusion dependency per visit and the presence of ring sideroblasts. When evaluating temporal changes in iron parameters, with linear quantile mixed models, we excluded patients from the timepoint they received iron chelation therapy. Overall survival (OS) was defined as the time from MDS diagnosis to death or, in case of progression-free survival, to date of progression or death; patients still alive at the end of follow-up were censored. Time-dependent Kaplan–Meier curves and cox proportional hazards models were used. In total, 256 consecutive patients, were included in this study. Over five six-monthly visits, 1040 samples were collected. Table 1 describes the patient characteristics. Most patients without ring sideroblasts were transfusion-independent at diagnosis (nonRS-TI; 55.9%), 18.8% with ring sideroblasts were transfusion-independent (RS-TI), 18.4% without ring sideroblasts were transfusion-dependent (nonRS-TD), and 7% with ring sideroblasts were transfusion-dependent patients (RS-TD). The median follow-up time was 6.6 years (95% CI 5.9–7.0). LPI was positively correlated with transferrin saturation (TSAT) (r = 0.15, p < 0.001, Fig. S1). LPI values increased exponentially at TSAT values above 80%. This effect was most pronounced in the transfusion-dependent groups, but also observed in the RS-TI group. MDA was weakly correlated with NTBI (r = 0.09, p = 0.069) and negatively correlated with hemoglobin level (r = −0.1, p = 0.033). GDF15 and hepcidin were negatively correlated in the RS-TI and nonRS-TD group and significantly negatively correlated in the RS-TD group (r = −0.34, p = 0.007, Fig. S2). Serum ferritin levels were elevated in all subgroups with a mean value of 858 µg/L at visit 5. The highest serum ferritin levels were observed in the RS-TD group (mean value at visit 5: 2092 µg/L, Table S1). Serum ferritin increased significantly per visit in the RS-TD group (beta 454.46 µg/L; 95% CI 334.65–574.27), but not in the other groups (Table S2). All subgroups, except for the nonRS-TI, had elevated TSAT levels. TSAT levels were most markedly increased in the RS-TD group with a mean TSAT of 88% at visit 5 (Table S1). In both transfusion-dependent groups the median increase per visit was significant (Table S2). LPI was elevated in the RS-TD group exclusively with a mean value of 0.59 µmol/L at visit 5 (Table S1). NTBI was elevated in all subgroups, with the highest values in the RS-TD group (Table S1). The increase in median NTBI level was significant in both transfusion-dependent groups (Table S2). Hepcidin levels were markedly elevated in the nonRS-TD group. Interestingly, hepcidin levels were lower in the RS-TD group, probably reflecting ineffective erythropoiesis, likewise supported by lower hepcidin/ferritin ratios in RS groups (Table S1). Median hepcidin levels increased over time in the transfusion-dependent subgroups only (Table S2). GDF15 levels, analyzed in the light of its potential role in hepcidin suppression, were increased in all subgroups (Table S1). The RS subgroups had higher GDF15 levels compared to the nonRS groups, reflecting increased erythropoiesis. Mean sTfR levels were within the reference range in all subgroups except for the RS-TI group, which showed elevated levels, reflecting...

Summary 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.

N, et al. Immunologically silent cancer clone transmission from mother to offspring. Proc Natl Acad Sci USA. 2009;106:17882–5. 7. Maruko K, Maeda T, Kamitomo M, Hatae M, Sueyoshi K. Transplacental transmission of maternal B-cell lymphoma. Am J Obstet Gynecol. 2004;191:380–1. 8. Osada S, Horibe K, Oiwa K, Yoshida J, Iwamura H, Matsuoka H, et al. A case of infantile acute monocytic leukemia caused by vertical transmission of the mother’s leukemic cells. Cancer. 1990;65:1146–9. 9. del Campo AB, Kyte JA, Carretero J, Zinchencko S, Mendez R, GonzalezAseguinolaza G, et al. Immune escape of cancer cells with beta2microglobulin loss over the course of metastatic melanoma. Int J Cancer. 2014;134:102–13. 10. McGranahan N, Rosenthal R, Hiley CT, Rowan AJ, Watkins TBK, Wilson GA, et al. Allele-specific HLA loss and immune escape in lung cancer evolution. Cell. 2017;171:1259–71.

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.

Introduction: CNS relapse of DLBCL is associated with poor prognosis. Estimated incidence varies between 1.9 and 8.4%1. The CNS-International prognostic index (IPI)2 help risk stratify and estimate the 2-year risk of CNS relapse in DLBCL patients treated with R-CHOP chemotherapy. CNS prophylaxis is indicated in patients with a high risk of CNS relapse (a score of ≥4 equated to a 10.2% risk). High-risk DLBCL patients outside the CNS-IPI system include double/triple-hit (MYC/BCL-2/BCL-6 translocations) lymphoma, HIV lymphoma, testicular lymphoma, primary cutaneous lymphoma-leg type, stage IE breast lymphoma3. IT methotrexate or cytarabine administered during the course of systemic chemotherapy has been the most widely employed method of CNS prophylaxis but there is paucity of data validating its efficacy. Aim: The primary aim of the study was to evaluate the CNS relapse rates in DLBCL patients who received CNS prophylaxis. Patients and Methods: This was a single-centre retrospective observational study conducted in a district general hospital. Data was extracted from the regional (Dorset Cancer Network) DLBCL database and laboratory reports for CSF analysis at the time of the first intrathecal chemotherapy. Medical records of patients with DLBCL who received CNS prophylaxis were evaluated for the following patient-related and disease-related demographics: age at diagnosis, gender, stage, systemic treatment, CNS prophylaxis, treatment response, remission duration, systemic relapse rates, CNS relapse rates and survival. CNS-IPI scores were retrospectively calculated and additional indications evaluated for patients who received CNS prophylaxis. Results: Between 2013 and 2018, 178 patients were diagnosed with DLBCL. All patients were treated with RCHOP chemo-immunotherapy. CNS prophylaxis was administered in 47 (26%) patients. Median age was 69 years (range 20-86 years) and 62% were males. All 47 patients (100%) received IT methotrexate as CNS prophylaxis, with 43 (91%) receiving all of the planned 4 doses of IT methotrexate 12.5 mg each. A CNS-IPI score of ³4 was present in 31 (66%) patients, and a score of 2-3 in 9 (19%) patients. Additional risk factors identified included testicular lymphoma in 3 patients, breast lymphoma in 2 patients and oropharyngeal lymphoma in 2 patients. Ten (21%) patients received their treatment at the outset with courses 1-4 of R-CHOP. Of the 47 patients who received CNS prophylaxis, 5 (10%) relapsed; all had isolated CNS lymphoma at relapse. Median time to CNS relapse was 25 months (range 12-36 months) from initial diagnosis of DLBCL. Median survival after CNS relapse was 5 months (range 2-9 months). Of the remaining 141 patients, 2 patients relapsed with isolated CNS lymphoma. Conclusion: Although the overall incidence was low (4%), CNS relapse was observed in 10% of high-risk patients all of whom received CNS prophylaxis with IT methotrexate. The efficacy of CNS prophylaxis with IT chemotherapy remains unproven. There is no randomised study to show that IT prophylaxis alone is effective. Current British guidelines recommend high-dose intravenous methotrexate over IT methotrexate if patient's physiological fitness and renal function are acceptable4. The median age in our cohort was 69 years which makes it challenging to deliver dose-intensive systemic therapy concurrently with intravenous high-dose methotrexate. The role of CNS prophylaxis in high-risk patients including its efficacy and safety in older patients need further evaluation in prospective randomised studies. References Eyre T et al.Efficacy of central nervous system prophylaxis with stand-alone intrathecal chemotherapy in diffuse large B-cell lymphoma patients treated with anthracycline-based chemotherapy in the rituximab era: a systematic review. Hematologica. 2019;105(7):1914-1924.Norbert Schmitz et al.CNS International prognostic Index: A risk model for CNS relapse in patients with diffuse large B-cell lymphoma treated with R-CHOPJ Clin Oncol 2016; 34:3150-3156.Andrew D Zelenetz et al.National Comprehensive Cancer Network (NCCN) Guidelines: B-Cell Lymphomas.Version 2.2020.Pamela McKay et al.The prevention of central nervous system relapse in diffuse large B-cell lymphoma: a British Society for Haematology good practice paper. Onlinelibrary.wiley.com. 2020. Available from: https://onlinelibrary.wiley.com/doi/epdf/10.1111/bjh.16866 Disclosures Hall: Janssen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings;Karyopharm:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings;Takeda:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings;Celgene:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored for educational meetings.Killick:Celgene:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending educational meetings;Jazz Pharmaceuticals:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending educational meetings;Novartis:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending educational meetings;Gilead:Honoraria, Other: Support for attending education meetings.McCarthy:Janssen:Honoraria;Abbvie:Membership on an entity's Board of Directors or advisory committees.Walewska:AbbVie:Other: sponsored for educational meetings, Speakers Bureau;Janssen:Other: sponsored for educational meetings, Speakers Bureau;Gilead:Speakers Bureau;Astra Zeneca:Membership on an entity's Board of Directors or advisory committees.Chacko:Astellas:Honoraria;Daiichi-Sankyo:Honoraria;Novartis:Honoraria, Other: Travel Grants;Gilead:Other: Travel grants;Jazz Pharmaceuticals:Other: Travel grants;Celgene:Other: Travel grants.

Background: CMML is a heterogeneous disease with overlapping features of MDS and MPN. Several risk scores have been proposed in CMML, including CPSS (Such Blood 2013) that identifies 4 risk groups (low, intermediate-1 [int-1], int-2 and high risk) with median overall survival (OS) ranging from 12 months to > 5 years. HSCT is the only curative treatment in CMML, with an expected 20-40% post-transplantation OS. Current guidelines, common to MDS and CMML recommend rapidly transplanting higher-risk CMML and delaying transplant in lower-risk disease (de Witte Blood 2017). Retrospective transplant studies have shown better outcome in lower-risk CMML (Liu et al BBMT 2017). Whether this translates into a survival benefit compared to a delayed transplant strategy requires prospective studies. Multistate models based on retrospective data from transplant and non-transplant patients (pts) have addressed similar issues in MDS (Koreth JCO 2013, Della Porta Leukemia 2017). Through a large collaborative study, we address the question of optimal timing for HSCT in CMML patients through a similar large international collaborative study. Method: We retrospectively selected pts from 2 registries: International MDS/MPN Working Group (IWG cohort, Padron Blood Cancer J 2015) and EBMT with the following criteria: WHO-defined CMML, age ≤ 70y, ECOG 0-2, diagnosis after 2000, available CPSS at the time of diagnosis. Different states were considered in higher- (CPSS int-2 and high) and lower- risk (CPSS low/int-1) pts: diagnosis, AML transformation, transplantation and death. IWG data was used to estimate transition probabilities from diagnosis to AML, transplantation and death and from AML to transplantation and death. EBMT data was used for transplantation to death transitions (Figure 1). Results: 719 and 403 pts were identified in the IWG and EMBT registry, respectively (resp). Median age was 64 (range 16-70) in IWG and 58 (19-70) years in EBMT cohort. Patients were male in 69% and 67% of IWG and EBMT cohorts, respectively (resp). CPSS was low in 22% and 13%, int-1 in 31% and 31%, int-2 in 40% and 45% and high in 8% and 11% of the IWG and EBMT pts, resp. In the IWG cohort, the 1 year cumulative incidence of transformation into AML were 7.3% and 18.2% in lower and higher risk patients, resp. Among the 719 pts from IWG, 102 received HSCT. At the time of diagnosis the expected life time of higher risk (int-2 and high) patients was 25.2 months while it was 44.1 months in lower risk (int-1 and low) patients. Table 1 reports gain or loss in expected life time (until 60 months after diagnosis) for transplantation at different intervals from diagnosis (within 6, 12, 18 and 24 months) for patients who have survived and not transformed to AML until the prediction time point, taking into account CPSS at diagnosis. Expected life time is also given separately for higher risk pts after transformation into AML. There was a modest gain of life expectancy with transplantation in higher risk patients (CPSS int-2 and high) while it was not the case in lower risk patients. The survival gain in higher risk pts with HSCT rose from 1.11 at 6 months to 3.4 months at 24 months. There was also a survival benefit of HSCT in higher risk pts who were transformed to AML during their follow-up which was even higher in terms of gain of months: from 9.89 month if HSCT performed within 6 months to 14.25 months if performed within 18 months. Of note, the loss of survival in lower risk patients who underwent HSCT decreased over time: 11.1 months if HSCT is performed within 6 months and 3.7 months if HSCT is performed within 24 months, probably owing to transition to higher-risk CMML at this stage in some pts, a transition that could not be captured in the present dataset. Conclusion: For the first time, using a multistate model, we could provide evidence that the transplant benefit in CMML is restricted to higher-risk pts, in line with findings in MDS and current guidelines. More analyses will be performed to analyze other subgroups of patients, especially regarding their risk (other classification than CPSS, the role of somatic mutation…). More studies are needed to analyze the role of pre HSCT treatment and its potential impact on post-transplant outcome. Disclosures Robin: Novartis Neovii: Research Funding. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria. Fenaux:Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Aprea: Research Funding. Kroeger:Riemser: Research Funding; Novartis: Honoraria, Research Funding; Medac: Honoraria; DKMS: Research Funding; Neovii: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; JAZZ: Honoraria; Sanofi-Aventis: Honoraria. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Finke:Riemser: Honoraria, Other: research support, Speakers Bureau; Neovii: Honoraria, Other: research support, Speakers Bureau; Medac: Honoraria, Other: research support, Speakers Bureau. Komrokji:Agios: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau; Incyte: Consultancy. Killick:JAZZ: Honoraria; Celgene: Honoraria; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; ALEXION: Honoraria. Blaise:Molmed: Consultancy, Honoraria; Sanofi: Honoraria; Pierre Fabre medicaments: Honoraria; Jazz Pharmaceuticals: Honoraria. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Background: There are limited treatment options for red blood cell (RBC) transfusion dependent (TD) LR (IPSS Low/Int-1) MDS patients who are relapsed/refractory to ESAs. Imetelstat is a first-in-class telomerase inhibitor that targets cells with short telomeres and active telomerase, characteristics observed in some MDS patients across all disease stages. Preliminary results show that imetelstat is effective treatment in LR-MDS patients inducing durable TI (Steensma et al ASH 2018 Abstr463). Aims: We report updated efficacy data with a median follow-up of 12.1 months in 38 LR non-del(5q) MDS patients, R/R to ESA and LEN/HMA naive from the open-label, single-arm Part 1 of IMerge, an ongoing phase 2/3 study (NCT02598661). Methods: Part 1 of the IMerge study included patients with LR MDS, who were heavily transfused (≥4U/8wks), were R/R to ESA or had sEPO >500 mU/mL. Imetelstat 7.5 mg/kg was administered IV every 4 weeks. The primary endpoint was 8-week TI rate; key secondary endpoints included 24-week TI rate, safety, duration of TI, and hematologic improvement (HI) rate. Among the initially enrolled patients, higher 8-week TI rate was observed in the non-del5q, LEN/HMA naive patients. Therefore, the study was amended to subsequently enroll only these patients. From a total of 57 patients enrolled in Part 1, 38 were non-del(5q), LEN/HMA naïve patients (13 in the initial and 25 in the expansion cohort). Here we report long-term efficacy, safety and biomarker data from these 38 patients. Results: Median baseline RBC transfusion burden was 8U/8weeks (range 4–14), 37% of the patients had IPSS Int-1; 71% had WHO 2001 RARS or RCMD-RS subtype and 32% with evaluable sEPO levels had baseline level >500 mU/mL. As of 23 January 2019, median follow-up was 12.1 months for the 38 patients, representing 30.4 and 11.6 months for the initial 13 and additional 25 patients, respectively. The 8-week TI rate was 45% (17/38) and median TI duration was 8.5 months (range 1.8–32.4). Of the 17 responding patients, 10 (59%) remained transfusion free for over 24 weeks. The 8-week TI rate did not differ based on the presence of ring sideroblasts or baseline sEPO levels. The 24-week TI rate was 26% (10/38). Erythroid HI, defined as transfusion reduction by at least 4 units /8 weeks (IWG2006), was achieved in 68% (26/38) of the patients. The most frequently reported adverse events were manageable and reversible grade ≥3 cytopenias. 6/38 patients had IPSS-R intermediate/poor cytogenetic risk. All 6 patents achieved 8-week TI; 2/6 patients achieved partial cytogenetic response. Post treatment decrease in telomerase hTERT RNA level was observed in 25/34 (73.5%) patients with available sample. Among 7 patients with pre- and post-treatment mutation analyses, six had SF3B1 mutations at baseline, and a decrease in the mutation VAF was observed in 2 patients that had longest TI duration on study. Summary/Conclusion: In high RBC transfusion burden patients with non-del(5q) LR-MDS R/R to ESA and naive to LEN/HMA, single-agent imetelstat yielded 8-week TI rate of 45%, with a median duration of 8.5 months (range 1.8–32.4). The 24-week TI rate was 26%. HI-E rate was 68%. All patients with IPSS-R intermediate and poor cytogenetic risk responded. Biomarker analyses of telomerase activity and mutation allele burden indicate an effect on the malignant mutant clone. These data support Part 2 of IMerge, Phase 3 placebo-controlled, randomized portion of the study, expected to open mid-2019.

Abstract Background Red cell transfusion is the most common intervention in the management of myelodysplastic syndromes (MDS). Transfusion practice in MDS is typically characterized by transfusion of multiple (usually 2 - 4) units every 3 - 4 weeks, but there is no supporting evidence for efficacy of this strategy. The aim of this trial was to explore the feasibility of (and adherence to) two standardized transfusion algorithms (liberal vs. restrictive), in order to inform future research. This trial is aligned with a Canadian study (NCT02099669). Methods The study was undertaken in the UK and Australia/New Zealand (ISRCTN26088319). Inclusion criteria were MDS patients with <20% bone marrow blasts, who were transfusion dependent (at least 1 red cell transfusion episode per month in the last 8 weeks) and receiving no additional MDS therapy. Randomization was to one of two red cell transfusion strategies over a 12-week period after a 4-week run-in to achieve a Hb >100 g/l prior to initiation of the allocated transfusion arm: (1) Restrictive (control arm) transfusion strategy to maintain hemoglobin concentration (Hb) between 85 and 100g/L; 2 units of packed red cell unit transfusions were transfused when Hb was <80g/L and 1 unit of packed red cell unit transfusions when Hb is 80-85g/L. (2) Liberal transfusion strategy to maintain Hb between 110 and 125g/L; 2 units of packed red cell unit transfusions were transfused when Hb was <105g/L and 1 unit of packed red cell unit transfusions when Hb is 105-110g/L. Primary outcomes were: percentage of pre-transfusion Hb concentrations below the target range of the assigned strategy, and achievement of at least a 20g/L difference between the mean pre-transfusion Hb in the two transfusion groups. It was pre-specified that the study would be considered feasible if compliance to the pre-transfusion target Hb was ≥70%, with evidence of a difference in Hb between the two arms. Secondary outcomes included quality of life questionnaires (EQ-5D-5L and EORTC QLQ-C30). The primary analysis was intention to treat and these results are reported. Results 38 patients were randomized from 12 hospitals (n=20, restrictive; n=18 liberal). Median age was 79 years (IQR 69-82). Although generally well matched, there were some minor imbalances in baseline characteristics (ECOG status, WHO subtype, prior iron chelation treatment and heart failure medication). Thirty-four participants received at least one transfusion during the trial and were included in the 'compliance to treatment threshold' analysis. Percentage compliance (95% confidence interval) of pre-transfusion Hb being below the target range of the RBC transfusion threshold assigned were 86% (75-94) and 99% (95-100) for the restrictive and liberal arms respectively. As compliance was ≥70% in both arms, the study was declared feasible. The mean (standard deviation (SD)) pre-transfusion Hb for the restrictive and liberal arms were 80 (6) g/l and 97 (7) g/l respectively, for a significant difference between the two groups (p<0.0001). Figure 1 shows the mean (±SD) Hb by week for all randomized participants by treatment arm. The total number of red cell units transfused was 82 in the restrictive group and 192 in the liberal group. Of 8 serious adverse events reported, 1 was deemed potentially related to transfusion. Compliance for completion of EQ-5D-5L and EORTC QLQ-C30 questionnaires was good (between 70 - 80%). Although interpretation of quality of life analyses is exploratory, the numbers of participants achieving a (pre-defined) clinically meaningful increase showed small improvements favoring the liberal policy across the following domains (EQ-5D-5L descriptive; EORTC QLQ-C30: physical functioning and global health score). Discussion These results of this multicenter trial in an older out-patient based population support the feasibility of progressing to a definitive trial of different red cell transfusion strategies, in order to evaluate the comparative effectiveness and safety for clinically relevant outcomes of a restrictive vs liberal transfusion policy for transfusion-dependent MDS. The large difference in units transfused between the two arms challenges perceived dogma and supports the need for a cost-effectiveness component in follow-on trials. (Funding: NHSBT, ANZSBT). Disclosures Buckstein: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

Background: Patients with lower-risk MDS (LR-MDS) are prone to iron toxicity due to long-term iron accumulation either caused by RBC transfusions or ineffective erythropoiesis. Nontransferrin bound iron (NTBI), including labile plasma iron (LPI), are toxic iron species that may mediate cellular damage via oxidative stress. The EUMDS registry collects prospective observational data on newly diagnosed LR-MDS patients from 145 centers in 17 countries since 2008. Methods: We analyzed serum from 247 LR-MDS patients collected at six-month intervals for ferritin, transferrin saturation (TSAT), hepcidin-25, soluble transferin receptor (sTfR) and toxic iron species (NTBI and LPI) in order to evaluate temporal changes in iron metabolism, the presence of potentially toxic forms of iron and their impact on survival, and quality of life. In addition, we measured the impact of iron chelation on the iron species levels and its impact on the outcome parameters. Results: The median age was 73 years (range: 37 to 95 years) and 66% were males. WHO2001 MDS-subtypes were RCMD (45%), RARS (22%), RA (18%), RAEB-1 (7%), 5q-syndrome (4%) and RCMD-RS (4%). The IPSS-R categories were: (very) low risk: 66%; intermediate risk: 11%; (very) high risk: 2% and unknown: 20%. The median EQ5D index score was 0.80 (p10 to p90: 0.52 to 1.00). The table shows iron parameters at registration, 1 and 2 years follow-up both in transfusion-dependent (TD) and transfusion-independent (TI) patients and according to: MDS-RS (RARS/RCMD-RS) or MDS Other (RA/RCMD/RAEB/5q-syndrome). Mean serum ferritin levels were increased in TD patients, compared to TI patients (Table). Increase of ferritin levels over time was high in both TD groups, but the increase was more pronounced in the RS subgroup. Elevated CRP levels (> 10mg/L) were observed in TD nonRS patients, especially during the first year after diagnosis. Markedly increased mean TSAT levels (>75%) occurred in the subgroup of TD-RS patients throughout the observation period. Hepcidin levels were most markedly elevated in TD nonRS patients at registration and remained elevated during follow-up (Table). Hepcidin levels were low in MDS-RS TI patients at all time points compared to nonRS MDS patients and decreased over time as a result of an increased (ineffective) erythropoiesis (Table). This is supported by the highest levels of STfR also noted in this patient category (data not shown). The highest NTBI and LPI levels were observed in TD-RS patients compared to the other 3 subgroups (Table). Both NTBI and LPI levels had a strong correlation (p <0.001) with TSAT. Elevated LPI levels in combination with high TSAT levels (>80%) occurred almost exclusively in patients with MDS-RS and/or previous transfusions. Both the EQ5D index score and EQ-VAS showed a negative correlation (r) with LPI levels with r = -0.09 (p = 0.028) and r = -0.07 (p=0.046) respectively. This negative effect of elevated LPI levels was most pronounced in the TD RS subgroup with a negative correlation of -0.17 for the EQ5D index score and -0.2 for the VAS score. In total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 4 patients desferioxamine and 1 patient unknown). LPI levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferoxamine treatment. The Kaplan-Meier curves (Figure) demonstrate the prognostic impact of elevated LPI levels by transfusion status as a time dependent variable; once a subject had an elevated LPI level, they remained in this group. Patients were censored at time of starting iron chelation (16 patients). In a multivariate analysis comparing elevated LPI levels and transfusion dependency to the control group with undetectable LPI and no transfusion showed a significantly decreased survival in all 3 risk groups after adjustment for age at diagnosis, baseline IPSS-R category and ESA treatment status; for details, see supplementary table. Conclusion: This study illustrates labile plasma iron species as a clinically relevant assay for identification of the toxic fraction of overt iron overload and its negative impact on HRQoL and overall survival in transfusion dependent and transfusion independent patients. Table. Table. Culligan: Merck Sharp & Dohme (MSD): Honoraria; Abbvie: Other: Support to attend conferences; Takeda: Honoraria, Other: Support to attend conferences; Pfizer: Honoraria; Celgene: Other: Support to attend conferences; JAZZ: Honoraria; Daiichi-Sankyo: Other: Support to attend conferences. Garelius:novartis: Honoraria. de Witte:Celgene: Honoraria, Research Funding; Novartis: Research Funding; Amgen: Consultancy, Research Funding.

Deletion of the long arm of chromosome 5 [del(5q)] is the most common cytogenetic abnormality found in the myelodysplastic syndromes (MDS).[1][1] Patients with the 5q-syndrome have macrocytic anemia and the del(5q) as the sole karyotypic abnormality.[1][1] Haploinsufficiency of the ribosomal protein

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.