Myelodysplastic Syndrome With Ring Sideroblasts Research Articles

Long-Term Clonal Inversion in an MDS-RS Case with Dual SF3B1 Mutations

Recent large-scale studies have traced SF3B1 mutations as a driving event in clonal establishment and expansion, and a hallmark of myelodysplastic syndrome with ring sideroblasts (MDS-RS). However, the mechanisms underlying the fitness advantage allowing SF3B1-mutant (SF3B1mt) clones to thrive remain poorly understood. Thus, tracking these events in conditions of low noise is crucial to our comprehension of clonal hematopoiesis. Here we report a 12-year clinical and molecular follow-up of a unique dual SF3B1mt MDS-RS case displaying a dynamic state of clonal competition between two clones propagated by a N626D and K666N mutation, respectively (Panel A). After an initial rapid expansion of the N626D clone compared to the relatively stable K666N clone during the initial follow-up period of ~2 years, the K666N clone spontaneously expanded at expense of the N626D clone to eventually dominate the bone marrow, driving a complete inversion of clonal prevalence. Clinically, these active shifts of high clonal involvement in the bone marrow were accompanied by stable subclinical anemia (median 12.5 g/L, range 12.0 - 13.2 g/L) and a continued decrease in the ring sideroblast phenotype over time (from 42% to 8% RS) in the absence of any treatment. The stability of this patient's clinical profile was therefore considered as unlikely to confound further characterization. Through a combination of whole-genome sequencing (WGS) of hematopoietic stem and progenitor cell (HSPC)-generated colony-forming units (CFU), combined DNA + RNAseq (TargetSeq) of FACS-purified hematopoietic stem cells (HSCs) and megakaryocyte-erythroid progenitor cells (MEP), and high-throughput 3'-based 10X Genomics single-cell RNAseq of CD34+ HSPCs, we molecularly characterized the two distinct SF3B1mt clones in bone marrow samples acquired before, during and after the points of clonal inversion (Panel B). Using WGS of single cell-derived colonies, we established that the acquisition of either the N626D or K666N mutation had preceded the diagnosis of MDS-RS by at least 20 years. Moreover, both mutations were associated with an increased mutational rate as compared to wildtype colonies, and the two clones presented distinct patterns of additional coding mutations as potential co-drivers of expansion. Mirroring previous characterizations of SF3B1mt HSPCs, we identified a common and deleterious alternative splicing profile to both genotypes via TargetSeq which included mis-splicing events in the known SF3B1mt targets MAP3K7 and SEPTIN6. In addition, we identified mutation-specific splicing profiles as potentially responsible for distinct downstream distinctions between both clones. Through this analysis, we identified the K666N-specific loss of expression of the tumor suppressors HOXA5, RGCC and NEURL1 as potential compound factors to the known higher risk underlying SF3B1K666N mutations. Finally, we disentangled clone-driven and aging-driven transcriptomic changes to identify a significant decrease of the early hematopoietic controllers ID1, ID2 and ID3 as a potential mechanism of aging-related cellular senescence. In conclusion, this case study comprises a unique long-term follow-up of the clonal dynamics underlying two co-existing, distinct and competing SF3B1mt clones which identifies subtle molecular changes and differences underlying the expansion and progression of SF3B1mt clones. *Moura PL, Hofman IJF, Nannya Y and Aliouat A contributed equally to this work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Prognostic Scoring Model for Myelodysplastic Syndrome Patients With SF3B1 Mutation.

The outcomes of myelodysplastic syndrome (MDS) patients with SF3B1 mutation, despite identified as a favorable prognostic biomarker, are variable. To comprehend the heterogeneity in clinical characteristics and outcomes, we reviewed 140 MDS patients with SF3B1 mutation in Zhejiang province of China. Seventy-three (52.1%) patients diagnosed as MDS with ring sideroblasts (MDS-RS) following the 2016 World Health Organization (WHO) classification and 118 (84.3%) patients belonged to lower risk following the revised International Prognostic Scoring System (IPSS-R). Although clonal hematopoiesis-associated mutations containing TET2, ASXL1 and DNMT3A were the most frequent co-mutant genes in these patients, RUNX1, EZH2, NF1 and KRAS/NRAS mutations had significant effects on overall survival (OS). Based on that we developed a risk scoring model as IPSS-R×0.4+RUNX1×1.1+EZH2×0.6+RAS×0.9+NF1×1.6. Patients were categorized into two subgroups: low-risk (L-R, score <= 1.4) group and high risk (H-R, score > 1.4) group. The 3-year OS for the L-R and H-R groups was 91.88% (95% CI, 83.27%-100%) and 38.14% (95% CI, 24.08%-60.40%), respectively (P<0.001). This proposed model distinctly outperformed the widely used IPSS-R. In summary, we constructed and validated a personalized prediction model of MDS patients with SF3B1 mutation that can better predict the survival of these patients.

Global Proteomics Analysis of Bone Marrow: Establishing Talin-1 and Centrosomal Protein of 55 kDa as Potential Molecular Signatures for Myelodysplastic Syndromes.

Myelodysplastic syndrome (MDS) is a hematological disorder characterized by abnormal stem cell differentiation and a high risk of acute myeloid leukemia transformation. Treatment options for MDS are still limited, making the identification of molecular signatures for MDS progression a vital task. Thus, we evaluated the proteome of bone marrow plasma from patients (n = 28) diagnosed with MDS with ring sideroblasts (MDS-RS) and MDS with blasts in the bone marrow (MDS-EB) using label-free mass spectrometry. This strategy allowed the identification of 1,194 proteins in the bone marrow plasma samples. Polyubiquitin-C (UBC), moesin (MSN), and Talin-1 (TLN1) showed the highest abundances in MDS-EB, and centrosomal protein of 55 kDa (CEP55) showed the highest relative abundance in the bone marrow plasma of MDS-RS patients. In a follow-up, in the second phase of the study, expressions of UBC, MSN, TLN1, and CEP55 genes were evaluated in bone marrow mononuclear cells from 45 patients by using qPCR. This second cohort included only seven patients from the first study. CEP55, MSN, and UBC expressions were similar in mononuclear cells from MDS-RS and MDS-EB individuals. However, TLN1 gene expression was greater in mononuclear cells from MDS-RS (p = 0.049) as compared to MDS-EB patients. Irrespective of the MDS subtype, CEP55 expression was higher (p = 0.045) in MDS patients with abnormal karyotypes, while MSN, UBC, and TALIN1 transcripts were similar in MDS with normal vs. abnormal karyotypes. In conclusion, proteomic and gene expression approaches brought evidence of altered TLN1 and CEP55 expressions in cellular and non-cellular bone marrow compartments of patients with low-risk (MDS-RS) and high-risk (MDS-EB) MDSs and with normal vs. abnormal karyotypes. As MDS is characterized by disrupted apoptosis and chromosomal alterations, leading to mitotic slippage, TLN1 and CEP55 represent potential markers for MDS prognosis and/or targeted therapy.

Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs

AbstractSF3B1K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1K700E- and SF3B1WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1K700E cells included, unexpectedly, motifs of the TEA domain (TEAD) transcription factor family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology.

Vitamin B5 and Succinyl-CoA Improve Ineffective Erythropoiesis in SF3B1 Mutated Myelodysplasia

Myelodysplastic syndrome (MDS) is a hematological clonal stem cell disease. Recurrent splicing factors mutations are reported in 50% of MDS. Interestingly, mutations in the splicing factor gene SF3B1 are over-represented in MDS with ring sideroblasts (MDS-RS), co-occurring in up to 90% of patients. In MDS-RS, anemia is the major clinical manifestation. Erythropoiesis stimulating agents (ESAs) are used to treat anemia; however, the overall response rates are 20% to 40% with a duration of response of 18-24 months. New therapeutic options are needed to improve response to ESAs treatment and delay red blood cell transfusion, which are associated with acute myeloid leukemia progression and increase in morbidity.Mutations in SF3B1 modify the recognition pattern of the 3' splice site and lead to subsequent mis-splicing of its targets. To identify critical mis-splicing events involved in the erythroid differentiation blockage, we performed splicing analysis on RNA sequencing generated from hematopoietic stem/progenitor cells undergoing differentiation. Three MDS primary samples harboring SF3B1 mutations and three age-matched healthy donors cultured under normoxia and hypoxia conditions were initially used for the analysis. High depth RNA sequencing and differential splicing analyses using rMATS identified 2,845 mis-spliced events including 200 shared between hypoxia and normoxia conditions. Here, using a cohort of 42 MDS samples, we report the mis-splicing of the coenzyme A synthase (COASY) transcript. Heme synthesis relies on succinyl-CoA synthesis, and its production itself depends on the availability of cellular CoA. We thus hypothesised that COASY mis-splicing is a key driver of ineffective erythropoiesis in MDS-RS patients.In primary hematopoietic cells, COASY is upregulated during erythroid differentiation and its silencing in CD34 + cells severely impedes the generation of mature erythroid cells CD71 - CD235a + and causes disruption in heme production. Functional characterisations of the CRISPR-CAS9 edited K562 SF3B1K700E and the SF3B1-mutated HNT-34 cell lines confirmed that COASY mis-splicing impairs COASY protein synthesis that ultimately results in 60% loss of the protein. Metabolomic analysis showed that COASY mis-splicing depletes cells in CoA and succinyl-CoA metabolites, however this phenotype can be rescued by supplementation with vitamin B5, a CoA precursor. Consequently, we showed in vitro that saturating the 40% of remaining COASY enzyme with vitamin B5 or supplementing medium with its downstream by-product, succinyl-CoA, improved erythropoietic differentiation in MDS SF3B1mut patients.In summary, our results for the first time show that SF3B1 mutations induce coenzyme A synthase (COASY) transcript mis-splicing, that consequently leads to measurable defects in metabolites essential for heme biosynthesis. Our report reveals a novel critical role of COASY in regulating normal bone marrow erythropoiesis through control of succinyl-coA during human erythroid differentiation. Remarkably, partial loss of the coenzyme A synthase in MDS-RS patients leads to disruption in the erythroid lineage as well as heme deficiency, that can be rescued by exogenous treatment with vitamin B5 or succinyl-CoA. Therefore, vitamin B5 could represent a very attractive agent to combine with existing treatments in order to increase erythroid maturation and delay red blood cell transfusion dependency in MDS-RS patients.Graphical representation: SF3B1 mutant causes mis-splicing in COASY that results in loss of protein. Deficiency in COASY triggers a downregulation of succinyl-CoA that is involved in the rate limiting step of heme synthesis. Heme deficiency subsequently impairs erythroid differentiation. Treatment of MDS SF3B1 mutant cells with vitamin B5 (precursor of CoA), or succinyl-CoA, rescues erythroid differentiation. [Display omitted] DisclosuresPlatzbecker: Geron: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; AbbVie: Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; StemLine: Consultancy; Takeda: Consultancy; Astex: Research Funding. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Isogenic MDS-RS Patient-Derived iPSCs Define the Mis-Spliced Transcript Repertoire and Chromatin Landscape of SF3B1-Mutant Hematopoietic Stem/Progenitor Cells

▪The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WT cells, recapitulating hallmark phenotypes of MDS cells.To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot.By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.), we found that the chromatin landscape of SF3B1K700E HSPCs resembled more this of megakaryocyte-erythroid progenitor cells (MEPs) and erythroid cells, whereas that of SF3B1WT HSPCs resembled more granulocyte-monocyte progenitors (GMPs) and monocytes. This finding may underlie the more prominent involvement of the erythroid lineage in the pathology and clinical presentation of MDS-RS. To interrogate transcriptional programs in SF3B1K700E mutant cells, we performed transcription factor (TF) motif enrichment analysis. Motifs enriched in ATAC-Seq peaks more accessible in SF3B1K700E cells that were linked to genes upregulated in SF3B1K700E cells, included motifs of several TFs with known roles in hematopoiesis (GATA, ETS, STAT, AP-1). Unexpectedly, motifs of the TEAD TFs were also enriched. The TEAD family of TFs are best known as effectors of the Hippo signaling pathway, with important roles in various biological processes and malignancies, albeit no clear links to adult hematopoiesis or hematologic disease. TEAD2 and TEAD4 were upregulated in SF3B1-mutant, compared to the WT, iPSC-HSPCs and TEAD transcriptional activity, measured with a luciferase reporter construct, was higher in SF3B1K700E, compared to SF3B1WT iPSC-HSPCs. We did not find expression or activation of YAP or TAZ, which bind to DNA as a complex with TEAD upon Hippo pathway activation. These results support a Hippo-independent increase of TEAD expression and activity in SF3B1K700E cells.In summary, we generated a panel of isogenic patient-derived iPSCs that allowed us to comprehensively characterize the transcriptome and chromatin landscape of SF3B1K700E HSPCs in an isogenic system, derive a splicing signature of SF3B1K700E and identify the TEAD TF as a new transcriptional regulator of SF3B1K700E mutant HSPCs.G. Asimomitis, A.G. Deslauriers: shared 1 st authorshipE. Papaemmanuil, E.P. Papapetrou: shared senior authorship DisclosuresDeslauriers: Novo Nordisk A/S: Current Employment. Hellström-Lindberg: Celgene: Research Funding. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Genomic and Transcriptomic Differences of Myelodysplastic Syndrome/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) and Myelodysplastic Syndrome with Ring Sideroblasts (MDS-RS)

INTRODUCTION: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare hematological disorder characterized by anemia, bone marrow dysplasia with ring sideroblasts and persistent thrombocytosis, and high frequency of SF3B1 and JAK2 mutations. Despite clinical, histological and molecular similarities with MDS with ring sideroblasts (MDS-RS), the clinical outcomes of these entities are diverse. To date, there is no data evaluating specific functional pathways which might explain phenotypic and clinical differences beyond diverse frequencies of JAK2 mutation. METHODS: We evaluated a total of 24 patients (pts) with MDS/MPN-RS-T and 27 pts with MDS-RS. Diagnosis was based on WHO 2017 criteria and confirmed by two independent hematopathologists. Whole bone marrow DNA was subject to 81 gene targeted next-generation sequencing (NGS) analysis. CD34+ cells from bone marrow samples of 4 pts with MDS/MPN-RS-T, 7 pts with MDS-RS and 17 healthy individuals obtained from AllCells (Emeryville, CA) were isolated using the CD34 MicroBead Kit and RNA was isolated using the PicoPure RNA isolation kit. Fastq files were mapped to the human genome (build GRCh38) in TopHat2 using the default options. Differential gene expression analysis was conducted using DESeq2 in R version 3.6.2. Pathway enrichment analysis was performed using gene set enrichment analysis, with the fgsea library in R. RESULTS: Patients with MDS/MPN-RS-T had higher median bone marrow ring sideroblast percentage (47% vs 32%, p=0.04) and absolute neutrophil count (4.34x109/L vs 2.99x109/L, p=0.001). Frequency of identified mutations and their VAFs compared to MDS-RS are shown in Figure 1A. The median number of mutations was higher in MDS/MPN-RS-T than in MDS-RS (3 vs 2, p&amp;lt;0.001). SF3B1 mutations were the most frequent in both entities (MDS/MPN-RS-T: 92%, MDS-RS: 82%), had similar median VAF (34% vs 32%, p=0.619), and involved the hot spot codon K700E in 64% and 43% of MDS-RS and MDS/MPN-RS-T (p=0.227), respectively. As expected, 58% of pts with MDS/MPN-RS-T had JAK2 V617F mutations but were also more likely to have mutations in kinase signaling genes (NF1, SETBP1, CBL, CBLB, FLT3 TKD, MPL) compared to MDS-RS (29% vs 4%, p=0.019). Four (40%) of JAK2 negative MDS/MPN-RS-T had mutations in kinase signaling genes. There were no differences in frequency of TET2 mutations between both entities. However, there was a trend for the median VAF of TET2 mutations in MDS/MPN-RS-T to be lower than in MDS-RS (1.5% vs 21.1%, p=0.177) suggesting a likely subclonal nature of these mutations compared to MDS-RS in which they appeared as dominant events. MDS/MPN-RS-T showed distinct transcriptomic profile compared to both healthy controls and MDS-RS. Compared to healthy controls, a total of 2 pathways were significantly upregulated and 58 were downregulated in MDS/MPN-RS-T while 5 pathways were upregulated and 69 were downregulated in MDS-RS. Compared to MDS-RS, a total of 29 pathways were significantly upregulated and 26 were downregulated in MDS/MPN-RS-T. The most significantly upregulated pathways in MDS/MPN-RS-T included genes involved in platelet activation and aggregation, cytokine signaling, and signaling through GPC receptors (Figure 1C). Compared to both healthy control and MDS-RS, MDS/MPN-RS-T was characterized by downregulation of genes involved in DNA damage response, regulation of apoptosis, telomere maintenance and RNA synthesis (Figure 1D). MDS-RS was characterized by downregulation of genes involved in signaling by GPC receptors and MAPK signaling, mRNA splicing, cytokine signaling and signaling through interleukins compared to both control and MDS/MPN-RS-T (Figure 1C). CONCLUSIONS: MDS/MPN-RS-T is characterized by co-dominance of SF3B1 and JAK2 mutations and presence of minor kinase signaling mutations not observed in MDS-RS. Upregulation of cytokine and interleukin signaling mediated through GPC receptors, and downregulation of genes involved in apoptosis and DNA damage are unique transcriptomic features of MDS/MPN-RS-T likely driven by genotype. These unique genomic and transcriptomic characteristics of MDS/MPN-RS-T supports the classification of MDS/MPN-RS-T based on genomic features beyond presence of SF3B1 mutation, and might represent potential therapeutic avenues for this rare disease. Figure Disclosures Sasaki: Otsuka: Honoraria; Pfizer Japan: Consultancy; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. Kantarjian:Sanofi: Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; Immunogen: Research Funding; Jazz: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Novartis: Honoraria, Research Funding; Delta Fly: Honoraria; Pfizer: Honoraria, Research Funding; Oxford Biomedical: Honoraria; Ascentage: Research Funding. Garcia-Manero:Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding; Onconova: Research Funding; Merck: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy.

Coordinated Mis-Splicing of Multiple Mitochondrial Iron Metabolism Genes Causes Ring Sideroblast Formation in SF3B1-Mutant MDS

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with &amp;gt;10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant &amp;gt;40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

Mutation Profiles Identify Distinct Clusters of Lower Risk Myelodysplastic Syndromes with Unique Clinical and Biological Features and Clinical Endpoints

Mutation Profiles Identify Distinct Clusters of Lower Risk Myelodysplastic Syndromes with Unique Clinical and Biological Features and Clinical Endpoints

Probing Aberrant Splicing in a Novel Model of SF3B1-Mutant Myelodysplastic Syndromes

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is widely appreciated to be a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous mutations in a core U2 spliceosome factor SF3B1 are strongly associated with MDS with ring sideroblasts (MDS-RS). MDS-RS is characterized by iron retention in mitochondria and ineffective erythropoiesis. How aberrant splicing induced by mutant SF3B1 causes ring sideroblasts and erythroid defects remains poorly understood. There are no genetically accurate models of MDS-RS that recapitulate ring sideroblast formation. Our lab has established an iPSC reprogramming approach to generate an in vitro model of MDS-RS. This method captures genetically distinct MDS subclones as well as normal cells in individual patients enabling internal normalization to the patient's isogenic normal clone. By differentiating iPSCs we generated inducibly immortalized MDS-RS CD34+ progenitor cell lines with robust expansion and erythroid differentiation potential enabling extensive analysis of SF3B1-mutant aberrant splicing throughout erythroid differentiation. We show that SF3B1-mutant lines form abundant ring sideroblasts compared to normal isogenic controls. We next performed RNA-sequencing and splicing analysis at different stages of erythroid differentiation. We show that global splicing patterns are maintained in SF3B1-mutant cells, while revealing distinct subsets of aberrantly spliced transcripts in CD34+ progenitors and erythroblast populations. This suggests that different aberrant splicing events contribute to erythroid progenitor expansion and inefficient erythropoiesis and iron dysregulation in MDS-RS. Taken together, this novel iPSC model of MDS-RS is a robust tool for studying the role of aberrant splicing in SF3B1-mutant erythroid progenitor expansion, inefficient erythropoiesis and ring sideroblast formation. Disclosures No relevant conflicts of interest to declare.

Marrow Ring Sideroblasts Are Highly Predictive for TP53 Mutation in MDS with Excess Blasts

Background: Myelodysplastic syndrome with ring sideroblasts (MDS-RS) is defined by the World Health Organization (WHO) as ring sideroblasts (RS) ≥15% or ≥5% with associated SF3B1 mutation and no excess blasts (EB). In MDS-RS, SF3B1 mutation defines a homogenous group with isolated erythroid dysplasia and favorable prognosis (Malcovati et al. Blood 2015). Given the separate WHO classification, patients with MDS-EB frequently are not tested for RS. SF3B1-wild type (wt) MDS with RS also has not been well characterized. Therefore, herein we characterized MDS with RS, focusing on SF3B1-wt and implications of molecular subsets. Patients and Methods: Between 2013 and 2018, 157 MDS and MDS/MPN patients with RS ≥5% and next generation sequencing performed within 6 months of diagnosis at Moffitt Cancer Center were identified with clinical variables obtained at date of diagnosis. Quantification of RS was performed in all cases by hematopathology. Baseline characteristics were compared by Fisher's exact test (categorical variables) and Mann-Whitney test (continuous variables). Survival estimates were calculated using the Kaplan-Meier method from date of diagnosis and groups were compared using log-rank test. Multivariate survival analysis performed by means of Cox proportional hazards regression. Pearson correlation coefficient was used in correlative analyses. Results: A total of 75 SF3B1-mutant (mt) and 82 SF3B1-wt cases with MDS (141) or MDS/MPN (13) and RS were identified. Median age was 71 years (38-89) with male and Caucasian predominance (62% and 94%, respectively). In the SF3B1-wt cohort, there were 77 MDS and 5 MDS/MPN patients. The MDS patients consisted of 2 MDS-SLD, 15 MDS-MLD, 3 MDS-RS-SLD, 17 MDS-RS-MLD, 24 MDS-EB1 and 16 MDS-EB2. The majority of SF3B1-wt patients (58%) were high or very high risk based on the Revised International Prognostic Scoring system (IPSS-R). Median RS% was significantly lower in SF3B1-wt compared to SF3B1-mt (18% (5-50) vs 35% (5-83) p &lt;0.0001). TP53 was the most common mutation (54%) in the SF3B1-wt cohort (n=44; Figure 1). Additional mutations observed in &gt;10% of the SF3B1-wt cohort were DNMT3A 18% (n=15), TET2 16% (n=13) and U2AF1 16% (n=13). Non-SF3B1 spliceosome mutations represented 27% (n=22) of the SF3B1-wt cohort. TP53-mt and non-SF3B1 spliceosome-mt were observed at significantly higher prevalence in SF3B1 wt vs mt patients (p&lt;0.0001 and p=0.003). In univariate analysis, IPSS-R, TP53 and DNMT3A were associated with worse overall survival in SF3B1-wt patients (OS). In multivariate analysis including age, IPSS-R and BMT, only TP53 was an independent covariate for inferior survival (HR 6.3; 95% CI 2.4-16.6 p&lt;0.0001). Given the high frequency of mutations, we then focused on TP53-mt RS patients. In the total cohort of patients with RS≥5%, 77% of MDS-EB1 and 68% of MDS-EB-2 were TP53-mt. In SF3B1 wt patients with RS≥5% and excess blasts, TP53 mutation was identified in 79% (n=19) and 81% (n=13) of MDS-EB-1 and MDS-EB-2 patients, respectively (p&lt;0.0001 TP53-mt MDS-EB vs other). 3 patients were co-mutant for TP53 and SF3B1. Increased RS as defined as &gt;15% vs 5-15% resulted in improved OS in the TP53-mt cohort (median OS 13.5 vs 8.6 months; HR 0.36 95% CI 0.14-0.93 p=0.034). In multivariate analysis including age, IPSS-R and BMT, the survival advantage was maintained (HR 0.35 95% CI 0.14-0.93 p=0.034). Increased RS did not significantly improve OS in any other somatic mutation. Response to hypomethylating agents was similar between TP53-mt RS &gt;15% vs 5-15% (Complete remission (CR) 21% vs 17% p=1.0 and overall response rate (ORR) 52% vs 42% p=.72). No TP53-mt RS patients responded to lenalidomide (0/4). The proportion of patients receiving allogeneic stem cell transplant was similar between TP53-mt RS &gt;15% vs 5-15% (14% vs 22% p=0.69). There was no difference in distribution of TP53-mt between 5-15% RS cohort vs &gt;15% (55% n=26 vs 51% n=18 p=0.82). Finally, TP53 VAF did not correlate with RS percentage (p=.393) Conclusions: In our study, MDS-RS-EB was highly concordant with the presence of TP53 mutation occurring in 80% of SF3B1-wt patients. In TP53 mutant patients, increased ring sideroblast % was an independent covariate associated with a significant survival advantage. As therapy targeting TP53 emerges, the ability to rapidly predict TP53 mutation status based on presence of ring sideroblasts should be a priority. Figure Disclosures Komrokji: Agios: Consultancy; celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Consultancy; Incyte: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sallman:Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding; Incyte: Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding, Speakers Bureau.

Characterization of Congenital Sideroblastic Anemia Model Due to ABCB7 Defects: How Do Defects in Iron-Sulfur Cluster Metabolism Lead to Ring Sideroblast Formation?

Backgroun d: The sideroblastic anemias (SAs) are a group of congenital and acquired bone marrow disorderscharacterized by bone marrow ring sideroblasts (RSs). The disease commonly presents as myelodysplastic syndrome with RS (MDS-RS), known as an acquired clonal SA that is strongly correlated with a specific somatic mutation inSF3B1 (splicing factor 3b subunit 1), which is involved in RNA splicing machinery. Thus far, several studies have consistently revealed compromised splicing and/or expression of ABCB7 (ATP-binding cassette subfamily B member 7) in MDS-RS harboring the SF3B1 mutation. ABCB7 encodes an ATP-binding cassette family transporter localizing to the inner mitochondrial membrane, and its loss-of-function mutation causes a syndromic form of congenital SA, which is associated with cerebellar ataxia. The substrates transported by ABCB7 are predicted to be iron-sulfur clusters (ISCs), which are essential for the function of multiple mitochondrial and extramitochondrial proteins, such as ferrochelatase and aconitase (its apo-form without ISC is called IRP1; iron regulatory protein 1). However, the detailed molecular mechanisms by which defects in ISC metabolism resulting from ABCB7 defects contribute to RS formation remains to be fully elucidated. Methods: Endogenous ABCB7 was depleted based on pGIPZ lentiviral shRNAmir (Dharmacon) in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells (Kurita et al., PLoS ONE, 2013). Puromycin (Sigma) was used for the selection of transduced cells. To induce terminal erythroid differentiation, HUDEP-2 cells were co-cultured with OP9 stromal cells (ATCC) in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum, erythropoietin, dexamethasone, monothioglycerol, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Fujiwara et al., MCB, 2019). For transcription profiling, Human Oligo Chip 25K (Toray) was used. Results: We first conducted ABCB7 knockdown in HUDEP-2 cells based on two independent shRNA plasmids. When the knockdown cells were induced to undergo erythroid differentiation,the majority of the erythroblasts exhibited aberrant mitochondrial iron deposition. Thus, we sought to clarify the potential causative link between ABCB7 defects and RS formation. Expression profiling revealed &gt;1.5-fold up- and down-regulation of 33 and 44 genes, respectively, caused by the ABCB7 knockdown. Intriguingly, 43% of the downregulated gene ensemble (19/44 genes) included multiple ribosomal genes, such as RPS2, RPL11,and RPS12. The downregulated genes also included HMOX1 (heme oxygenase 1), implying that heme biosynthesis would be compromised by the knockdown. Gene ontology (GO) analysis revealed significant (p&lt; 0.01) enrichment of genes associated with nuclear-transcribed mRNA catalytic process, cytoplasmic translation, and cellular iron ion homeostasis. Whereas the mRNA expression for ALAS2 (erythroid-specific 5-aminolevulinate synthase), encoding a rate-limiting enzyme of heme biosynthesis and one of the responsible genes for congenital SA, was not affected, its protein expression was noticeably decreased by ABCB7 knockdown, indicating that compromised transport of ISC from mitochondria to the cytosol may result in decreased ALAS2 translation by the binding of IRP1 to the iron-responsive element located in the 5'-UTR of ALAS2 mRNA.We are currently conducting detailed biological analyses to elucidate the causative link between defects in ISC metabolism due to ABCB7 defects and RS formation. Conclusion: We have first demonstrated the emergence of RS by ABCB7 depletion in human erythroblasts. Further characterization of the established SA model would aid in the clarification of its molecular etiology and the establishment of novel therapeutic strategies. Furthermore, our results may lead to a better understanding of the role of ISC in affecting cerebellar symptoms. Disclosures Fukuhara: Gilead: Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; AbbVie: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Bayer: Research Funding; Celgene Corporation: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Solasia Pharma: Research Funding. Onishi:Novartis Pharma: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa-Hakko Kirin: Honoraria; Celgene: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Clinical features and gene mutation spectrum in children with sideroblastic anemia

To study the clinical features and gene mutation spectrum of children with sideroblastic anemia (SA) and the clinical value of targeted next-generation sequencing in the molecular diagnosis of children with SA. Clinical data were collected from 36 children with SA. Targeted next-generation sequencing was used to detect mutations in SA-related pathogenic genes and genes associated with heme synthesis and mitochondrial iron metabolism. The association between genotype and clinical phenotype was analyzed. Of the 36 patients, 32 had congenital sideroblastic anemia (CSA) and 4 had myelodysplastic syndrome with ring sideroblasts (MDS-RS). Mutations in CSA-related genes were detected in 19 children (19/36, 53%), among whom 9 (47%) had ALAS2 mutation, 4 (21%) had SLC25A38 mutation, and 6 (32%) had mitochondrial fragment deletion. No pathogenic gene mutation was detected in 4 children with MDS-RS. Among the 19 mutations, 89% (17/19) were known mutations and 11% (2/19) were novel mutations. The novel mutation of the ALAS2 gene c.1153A>T(p.I385F) was rated as "possibly pathogenic" and the novel mutation of the SLC25A38 gene c.175C>T(p.Q59X) was rated as "pathogenic". ALAS2 and SLC25A38 gene mutations are commonly seen in children with CSA, but mitochondrial gene fragment deletion also accounts for a relatively high proportion. For children with hypoplastic anemia occurring in infancy, mitochondrial disease should be considered.

Germline and Acquired Genetic Variants in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction

Background and Aim:It is increasingly recognized that patients with a de novomyelodysplastic syndrome (MDS) onset as young adults, lacking any other feature of a congenital disorder, may share a pathogenic overlap due to the presence of both germline and somatic variants. Identifying an inherited pathogenic variant has important therapeutic implications beyond family counselling: adapting the selection of sibling donor, the use of highly cytotoxic therapy and the monitoring for other cancer development. However, most studies have focused on patients with suspected inherited disorders based on the presence of physical abnormalities and/or family history. In addition, a mixture of pediatric and adult cases is usually reported. The aim of this study is to characterize the germline and tumor variants in a group of adult MDS patients without accompanying congenital physical anomalies and or family antecedent of bone marrow failure.Methods: We included 72 patients from 15 Spanish centers with a diagnosis of MDS between 18 and 60 years old (y.o). Patients with a previously diagnosed or suspected (one physical anomaly or family history) congenital syndrome were excluded. Diagnoses were made in accordance with the WHO 2016 classification. Whole-exome sequencing (WES) libraries were prepared using SureSelectXT Target Enrichment and sequenced on a HiSeq4000 platform (IlluminaInc.). Mean number of reads per sample was 138,726,017 with a Phred Quality Score >30 in 95.05% of bases. Read mapping sequence alignment and variant calling were performed using Biomedical Workbench (Qiagen). WES was performed on 72 tumor and 32 paired germinal DNA (buccal swab). To identify potential germline-causal mutations, a selection tool was implemented incorporating 239 genes associated with cause or predisposition to bone marrow failure or cancer. Variants with an ExAC, TOPMed and/or European 1000 Genomes minor allele frequency ≥0.01 were discarded.Results: The median age at diagnosis was 49 y.o. The cohort was categorised into two groups, less or equal 50 y.o. (62.5%) and between 50 and 60 y.o. (37.5%). In the first group, the frequency according to the WHO classification were 12% MDS with single lineage dyplasia (MDS-SLD), 8% MDS with ring sideroblasts (MDS-RS), 11% MDS with multilineage dyplasia (MDS-MLD), 24% MDS with excess blasts(MD-EB), 4% MDS with isolated del(5q)(MDS-del5q), 4% MDS unclassifiable and 4% chronic myelomonocytic leukemia (CMML). Meanwhile, in the group with age more than 50 y.o., the subtypes were 3.7% MDS-SLD, 7.4% MDS-RS, 29.6% MDS-MLD, 40.7% MD-EB, 3.7% MDS-del5q, and 14.8% CMML.Patients less or equal 50 y.o. were stratified based on IPSS-R as very low (4%), low (64%), intermediate (20%), high (12%) and very high (0%); and the group of more than 50 y.o. as very low (14.8%), low (33.3%), intermediate (29.6%), high (11.1%) and very high (11.1%).The mean number of somatic mutations was 0.68 in patients with less or equal 50 y.o. and 1.37 in those between 50 and 60 y.o., p=0.033 (U Mann-Whitney); and regarding germline variants, the first group mean number was 2.44 (p25-75, 1-3) and the second group showed a mean of 1.85 (QI 25-75, 1-3), p= 0,331.In the whole cohort, germline variants were found in 62 out of 72 patients, with the following frequencies: ATR(N=5, 6.9%), followed by BARD1(N=5, 6.9%), ERCC6L2(N=4, 5.6%), MSH6(N=4, 5.6%), TCIRG1(N=4, 5.6%), NBEAL2(N=4, 5.6%), ASXL1(N=3, 4.2%), ATM(N=3, 4.2%), MPL(N=3, 4.2%), NF1(N=3, 4.2%), RECQL4(N=3, 4.2%), SAMD9L(N=3, 4.2%), WRN(N=3, 4.2%).Among germline variants, those reported previously as pathogenic or likely pathogenic, or involving genes associated with familial MDS/AML included: ERCC6L2(N=4, 5.6%), SAMD9L(N=3, 4.2%), and one case mutated for DDX41, FANCC, JAK2, MSH6, SETBP1, MUTYH, BRCA1and RECQL4. In the whole cohort, somatic variants were found in 38 patients, with the following frequencies: TP53(N=7, 9.7%), ASXL1(N=7, 9.7%), SETBP1(N=5, 6.9%), NF1(N=5, 6.9%), SRSF2(N=4, 5.5%).Conclusion:In this subset of young adults with de novo MDS without congenital anomalies and/or familial history suggesting the presence of an undiagnosed congenital syndrome, 18% of the cohort harbored a likely causative germline variant. In addition, we noted a predominance of variants affecting genes with a cancer predisposition limited to the hematopoietic system, rather than classical telomere, DNA damage genes with an established mendelian link. [Display omitted] DisclosuresDíez-Campelo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

A Leukemic Stem Cell Signature-Based Scoring System for Predicting Prognosis in Myelodysplastic Syndrome Patients

Leukemic stem cells (LSCs) possess biological properties shared with normal hematopoietic stem cells. They are responsible for chemoresistance and relapse in acute myeloid leukemia (AML). Although myelodysplastic syndrome (MDS) has traditionally been regarded as a “hematopoietic stem cell disorder”, the clinical and biological impact of LSCs on MDS patients are not well defined. To address this question, we used the Affymetrix HTA 2.0 microarray platform to profile 16 out of the 17 recently reported stemness genes (one of them, the ARHGAP22 gene was not included in our array) in our 160 adult primary MDS patients. The diagnoses were based on the 2016 World Health Organization (WHO) classification. Patients with antecedent chemotherapy or hematologic malignancies were excluded. Forty-one (25.6%) patients had MDS with single lineage dysplasia (MDS-SLD), 20 (12.5%) had MDS with ring sideroblasts (MDS-RS), 31 (19.4%) had MDS with multilineage dysplasia (MDS-MLD), 32 (20%) had MDS with excess blasts-1 (MDS-EB1) and 36 (22.5%), MDS-EB2. The risk distribution of the cohort was very-high risk, 15.3%; high risk, 21.3%; intermediate risk, 24.7%; low risk 34.7%; and very-low risk 4% according to the revised international prognosis scoring system (IPSS-R). We identified 4 genes (LAPTM4B, NGFRAP1, NYNRIN, and EMP1) whose expression levels were significantly correlated with overall survival (OS). An LSC4 score (0.731 x LAPTM4B - 1.259 x NGFRAP1 + 0.304 x NYNRIN + 0.231 x EMP1) was constructed based on the weighted sums derived from Cox regression analysis. Higher LSC4 scores were associated with higher IPSS-R scores, complex cytogenetics, and higher incidences of mutations of RUNX1, ASXL1, TP53, SRSF2 and ZRSR2. High-score patients had significantly higher 3-year AML transformation rate (36.3% vs 11.3%, P<0.001), shorter leukemia-free survival (LFS, median, 33.6 months vs not reached, P< 0.001, Figure 1A), and shorter OS (median, 15.9 months vs 77.3 months, P<0.001, Figure 1B). In subgroup analysis, LSC4 score could also well stratify MDS patients with normal karyotype into different risk groups (p<0.001). The prognostic significance of LSC4 scores for LFS, AML transformation rate, and OS still remained valid in both IPSS-R lower-risk (very low, low, and intermediate risk) subgroup (Figures 2A and 2B) and higher-risk subgroup (high and very high risk). In multivariate analysis, higher LSC4 score was an independent adverse risk factor for OS, irrespective of age, IPSS-R, and mutation status of ASXL1, TP53, EZH2, SRSF2, and ZRSR2 (Table). We also well validated the prognostic significance of the LSC4 scores in an independent cohort of 30 MDS patients (Figures 3A and 3B). Interestingly, patients with higher LSC4 scores had a better OS if they received allogeneic transplantation than those who did not. It implied that transplantation might ameliorate the poor survival impact of high LSC4 scores. In conclusion, through comprehensive analysis, we have created a simple and powerful LSC4 scoring system, which serves as an independent prognostic factor for OS and LFS in MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ineffective erythropoiesis in myelodysplastic syndrome

Ineffective hematopoiesis is one of the hallmarks of myelodysplastic syndrome (MDS). Recently, several signaling pathways responsible for inefficient erythropoiesis in MDS have been uncovered. The p53-S100a8/S100a9-TLR4 pathway is involved in ineffective erythropoiesis in 5q minus (5q-) syndrome. Somatic mutations target multiple components of the messenger RNA (mRNA) splicing machinery including Splicing Factor 3 Subunit b1 (SF3b1) and Serine Arginine Rich Splicing Factor 2 (SRSF2) in patients with MDS. SF3b1 is the most frequently mutated spliceosome component in MDS and is mutated approximately 85% of the time in MDS with ring sideroblasts (MDS-RS). SF3b1 mutations are not simple loss-of-function or inactivating mutations but result in a change of function and cause aberrant splicing of genes that may be involved in the pathogenesis of MDS-RS. Recurrent mutations are also found in epigenetic regulator genes in MDS, including polycomb repressive complex 2 (PRC2) genes. The loss of enhancer of zeste homolog 2 (EZH2), an enzymatic component of PRC2 in mice, markedly accelerates the development of MDS in combination with representative driver mutations. The loss of EZH2 causes impaired erythropoiesis and increases ineffective hematopoiesis. Of interest, EZH2 is one of the targets of SRSF2 mutants in MDS, and mis-spliced EZH2 mRNA undergoes nonsense-mediated decay (NMD) -dependent degradation. All these data suggest that EZH2 insufficiency causes ineffective erythropoiesis.

SF3B1-initiating mutations in MDS-RSs target lymphomyeloid hematopoietic stem cells

AbstractMutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal (SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro–B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS.

A Rare Case of Myelodysplastic Syndrome with Ring Sideroblasts, SF3B1 and TET2 Mutations in a Patient with Beta Thalassemia Trait

Concurrent myelodysplastic syndrome (MDS) and β-thalassemia trait is rare. We reported a case of a 59-year-old man with a known history of β-thalassemia (presumed to be β-thalassemia intermedia) presenting with progressive anemia and worsening fatigue. β-globin mutation analysis revealed a heterozygous mutation in the beta-globin gene, compatible with β-thalassemia trait. Bone marrow biopsy showed erythroid hyperplasia and erythroid dysplasia with increased ring sideroblasts (>15%). Additionally, there is also mild myeloid and megakaryocytic dysplasia. Molecular analysis revealed SF3B1 and TET2 mutations. These findings are consistent with myelodysplastic syndrome with ring sideroblasts (MDS-RS). To the best of our knowledge, this is the first report of MDS-RS with SF3B1 and TET2 mutations in a patient heterozygous for β-globin gene mutation. For β-thalassemia patients with worsening anemia, a comprehensive bone marrow analysis including cytogenetic and molecular studies is important to help further delineate the diagnosis. [N A J Med Sci. 2017;10(1):32-35. DOI: 10.7156/najms.2017.1001032]

Biology of sideroblastic anemia.

Sideroblastic anemia is characterized by anemia with ring sideroblasts produced by the bone marrow. Sideroblasts are formed by disutilization and deposit of iron in the mitochondoria. There are two forms of sideroblastic anemia: congenital and acquired. Congenital sideroblastic anemia is caused by mutations in genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, or mitochondrial metabolism. Although there is a variation in the mutated genes among races, the most common congenital sideroblastic anemia is X-linked sideroblastic anemia caused by mutations in the erythroid-specific δ-aminolevulinate synthase gene, which is the first enzyme of heme biosynthesis in erythroid cells. The most commonly acquired sideroblastic anemia is myelodysplastic syndrome with ring sideroblasts (MDS-RS). It has been shown that the splicing factor 3b subunit 1 (SF3B1) gene, which is a core component of the RNA splicing complex, is highly mutated in MDS-RS, although the underlying mechanism of the onset of the disease by the mutation of the SF3B1 gene remains unclear. Molecular analysis will contribute to the development of effective treatment for congenital and acquired sideroblastic anemia, which are intractable diseases.

Considering Bone Marrow Blasts from Nonerythroid Cells Improves the Prognostic Evaluation of Myelodysplastic Syndromes with Ring Sideroblasts

Introduction: As showed in a recent study of our group, considering bone marrow (BM) blasts from nonerythroid cellularity (NECs) improves the prognostic evaluation of MDS (Arenillas et al, J Clin Oncol 2016). By enumerating blasts from NECs, 12% of MDS patients diagnosed within WHO categories with less than 5% BM blasts were reclassified into higher-risk categories and showed a poorer overall survival than did those who remained in the initial categories. Refractory anemia with ring sideroblasts (RARS) and refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS) have shown an special good outcome in different studies. As MDS with ring sideroblasts (MDS-RS) usually present a high percentage of BM erythroblasts, considering BM blasts from NECs could imply a risk overestimation of this subset of patients.Aim: we evaluated the relevance of considering BM blasts from NECs or from total nucleated cells (TNCs) on classification and prognostication of the group of patients diagnosed with MDS-RS.Methods: We retrospectively analyzed 3,924 de novo MDS diagnosed according to WHO 2001 and 2008 classifications from the MDS Spanish registry. 1,045 patients presented less than 5% BM blasts from TNCs and equal or greater than 15% BM ring sideroblasts, fulfilling current definition for RARS (WHO 2001 and 2008) and RCMD-RS (WHO 2001). Moreover 1,233 patients with equal or greater than 5% BM ring sideroblasts and less than 5% BM blasts were analyzed in order to explore the future definition of WHO 2016, that considered as MDS-RS those patients with 5%-<15% BM ring sideroblasts if SF3B1 mutation is present. This was a tentative analysis since SF3B1 mutation information was not available in our series. Percentage of BM blasts from NECs was calculated as follows: [%BM blasts from TNCs/(100 - %BM erythroblasts) x 100]. Survival curves were constructed by using the Kaplan-Meier method and compared using the log-rank test. Univariable and multivariable Cox regression analysis were also implemented.Results: Median age at diagnosis of MDS-RS was 76y (25-101) and 59% were males. Estimated median follow-up, as calculated by reverse Kaplan-Meier method, was 50.1 months (95% CI, 45.5-54.7) and median OS was 96.5 months. By enumerating blasts from NECs, 10% of MDS-RS patients were reclassified into categories with equal or greater than 5% BM blasts and showed a poorer overall survival (OS) than did those who remained in initial categories (median OS, 68.1 vs 97.6 months, P=0.025; Hazard ratio (HR): 1.41; 95%CI: 1.04-1.91; p=0.026). After adjusting the survival analysis by IPSS cytogenetic risk groups, the prognostic impact of BM blasts considered from NECs maintained its significance (HR: 1.37; 95%CI: 1.01-1.85; p=0.045). Similar results were observed applying this method to the group of MDS patients with equal or greater than 5% BM ring sideroblasts and less than 5% BM blasts. By considering blasts from NECs, 10% of this subset of patients were reclassified into categories with equal or greater than 5% BM blasts and showed a poorer OS than did those who remained in initial categories (median OS, 60.2 vs 85.8 months, P=0.003; HR: 1.51; 95%CI: 1.15-1.97; p=0.003; HR adjusted for IPSS cytogenetics: 1.46; 95%CI: 1.12-1.92; p=0.006).Conclusion: considering bone marrow blasts from nonerythroid cells improves the prognostic evaluation of MDS with ring sideroblasts.Written on behalf of the Grupo Español de Síndromes Mielodisplasicos (GESMD). DisclosuresNo relevant conflicts of interest to declare.