Presence Of Ring Sideroblasts Research Articles

Treatment of Anemia in Lower-Risk Myelodysplastic Syndrome.

A majority of patients with lower-risk myelodysplastic syndrome (MDS) will present with or develop anemia. Anemia in MDS is associated with decreased quality of life and may correlate with decreased progression-free survival and overall survival. In this state of the art review we summarize current risk stratification approaches to identify lower-risk MDS (LR-MDS), the natural history of the disease, and meaningful clinical endpoints. The treatment landscape of LR-MDS with anemia is also rapidly evolving; we review the role of supportive care, erythropoietin stimulating agents, lenalidomide, luspatercept, hypomethylating agents (HMAs), and immunosuppressive therapy (IST) in the management of LR-MDS with anemia. In patients with deletion 5q (del5q) syndrome lenalidomide has both efficacy and durability of response. For patients without del5q who need treatment, the management approach is impacted by serum erythropoietin (EPO) level, SF3B1 mutation status, and ring sideroblast status. Given the data from the Phase III COMMANDS trial, we utilize luspatercept in those with SF3B1 mutation or ring sideroblasts that have an EPO level < 500 U/L; in patients without an SF3B1 mutation or ring sideroblasts there is equipoise between luspatercept and use of an erythropoietin stimulating agent (ESA). For patients who have an EPO level ≥ 500 U/L or have been previously treated there is not a clear standard of care. For those without previous luspatercept exposure it can be considered particularly if there is an SF3B1 mutation or the presence of ring sideroblasts. Other options include HMAs or IST; the Phase III IMERGE trial supports the efficacy of the telomerase inhibitor imetelstat in this setting and this may become a standard option in the future as well.

Early-Onset Myelodysplastic Syndromes (MDS) with Ring Sideroblasts (RS) without SF3B1 Mutations in Adults: Enrichment with Germline Variants in Genes Responsible for Congenital Sideroblastic Anemias

Introduction: Acquired mutations in SF3B1 gene in myeloid neoplasms are classically associated with the presence of RS in Pearl Stain, ineffective erythropoiesis, and favorable prognosis. Nevertheless, in up to 20-25% of adults diagnosed with MDS with RS (WHO 2017), mutations in SF3B1 are not found, and molecular grounds for the presence of RS remain to be ascertained. The objective of our study was to investigate whether the presence of RS could be associated with germline variants in genes responsible for congenital sideroblastic anemia (CSA). Methods: Patients diagnosed with de novo MDS between 16-60 years of age without previous organ dysfunction were recruited from 32 centers of GEMSD since 2016. Whole exomes were sequenced using HiSeq4000-NovaSeq6000-Illumina, paired tumor-germline samples. Mean depth was 100x, with 150 million reads per sample and quality Q30a&amp;gt;95%. Variants were analyzed using a bioinformatics pipeline: filtering intronic and synonymous variants and those with a population frequency &amp;gt;1%. The mutational state of SF3B1 was determined by Sanger Sequencing and Next Generation Sequencing (NGS). Germline variants were categorized according to American College of Molecular Genetics (ACMG) criteria. The list of genes related to CSA explored in this study is shown in Table 1. Results: Among 239 cases of adults diagnosed with early-onset MDS (mean age at diagnosis: 48 years, range 16-60), 58 (24%) patients presented with RS in bone marrow (mean RS: 28%). Of these 58 patients, acquired mutations in SF3B1 were not found in 32 (55%). Nine out of these 32 (25%) harbored a germline variant (two variants in one case) in genes responsible for CSA (Table 2): SLC25A38 (n=2), STEAP3 (n=2) FECH, ALAS2, GLRX5, SLC19A2, TRNT1 and IARS2. This frequency was statistically higher than in the SF3B1 and RS mutated group (n=23), with only one case with a germline variant in a CSA gene (p=0.013). It was also higher than in the non-RS cases (n=181), with only one case in this group (p&amp;lt;0.001). Using the Fisher's exact test, commonly used to perform enrichment, the odds ratios were also significant (p=0.033 and p&amp;lt;0.0001), respectively. Among patients with RS, those carrying a CSA gene germline variant were younger (43 vs. 54 years, p=0.04), had a higher rate of neutropenia (1.8 vs. 2.6 x 10E9/L, p=0.02), and thrombocytopenia (151 vs. 259 x 10E9/L, p=0.03) than MDS-RS patients with SF3B1 mutated. Furthermore, MDS-RS with a germline variant in CSA genes had a lower mean percentage of RS than patients who acquired the mutation in SF3B1 (14% vs. 38%; p=0.003). Conclusions: In our series, the frequency of MDS-RS without SF3B1 mutations is higher in early-onset adult MDS than the one reported in MDS in advanced age. Whole exome analysis allowed us to describe, for the first time, a significant enrichment of variants in genes causing CSA in young adults with RS and without acquired mutation in SF3B1.

Causes and Pathophysiology of Acquired Sideroblastic Anemia.

The sideroblastic anemias are a heterogeneous group of inherited and acquired disorders characterized by anemia and the presence of ring sideroblasts in the bone marrow. Ring sideroblasts are abnormal erythroblasts with iron-loaded mitochondria that are visualized by Prussian blue staining as a perinuclear ring of green-blue granules. The mechanisms that lead to the ring sideroblast formation are heterogeneous, but in all of them, there is an abnormal deposition of iron in the mitochondria of erythroblasts. Congenital sideroblastic anemias include nonsyndromic and syndromic disorders. Acquired sideroblastic anemias include conditions that range from clonal disorders (myeloid neoplasms as myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts) to toxic or metabolic reversible sideroblastic anemia. In the last 30 years, due to the advances in genomic techniques, a deep knowledge of the pathophysiological mechanisms has been accomplished and the bases for possible targeted treatments have been established. The distinction between the different forms of sideroblastic anemia is based on the study of the characteristics of the anemia, age of diagnosis, clinical manifestations, and the performance of laboratory analysis involving genetic testing in many cases. This review focuses on the differential diagnosis of acquired disorders associated with ring sideroblasts.

When Ring Sideroblasts on Bone Marrow Smears Are Inconsistent with the Diagnosis of Myelodysplastic Neoplasms.

Ring sideroblasts are commonly seen in myelodysplastic neoplasms and are a key condition for identifying distinct entities of myelodysplastic neoplasms according to the WHO classification. However, the presence of ring sideroblasts is not exclusive to myelodysplastic neoplasms. Ring sideroblasts are as well either encountered in non-clonal secondary acquired disorders, such as exposure to toxic substances, drug/medicine, copper deficiency, zinc overload, lead poison, or hereditary sideroblastic anemias related to X-linked, autosomal, or mitochondrial mutations. This review article will discuss diseases associated with ring sideroblasts outside the context of myelodysplastic neoplasms. Knowledge of the differential diagnoses characterized by the presence of ring sideroblasts in bone marrow is essential to prevent any misdiagnosis, which leads to delayed diagnosis and subsequent management of patients that differ in the different forms of sideroblastic anemia.

Recent advances in the knowledge of sideroblastic anemia

Sideroblastic anemias (SAs) are a group of heterogeneous congenital and acquired disorders characterized by anemia and presence of ring sideroblasts in the bone marrow. Congenital SA is a rare condition caused by mutations of genes involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. SAs can also occur following exposure to certain drugs or alcohol or caused by copper deficiency (secondary SA). SAs have been found to be associated with myelodysplastic syndrome (idiopathic SA), which strongly correlates with specific somatic mutations in SF3B1 (splicing factor 3b subunit 1), involved in the RNA splicing machinery. The recent widespread use of genome-editing technology and next-generation sequencing has led to a better understanding of the molecular pathophysiology of SAs. This review discusses the current understanding of the pathophysiology of SAs.

Luspatercept in MDS with Ring Sideroblasts: A Real-Life Multicenter Experience from a Named Patient Program

BackgroundLow risk Myelodysplastic syndromes (LR MDS) are a heterogeneous group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis, peripheral cytopenias and relative increase of bone marrow blasts [List 2004]. The most common cytopenia is anemia, which occurs in 85-90% of cases (isolated in 35% of cases) [Fenaux 2013], and ranges from mild/asymptomatic to transfusion dependent. The standard of care for anemia remains supportive care and about 70% of cases benefits from erythropoiesis-stimulating agents (ESAs).According to the FAB classification, a low-risk group of MDS characterized by the presence of ring sideroblasts (RS) was recognized. In the 2017 WHO classification, the nosologic entities of MDS uni-linear and multi-linear dysplasia with RS (MDS-UD-RS, MDS-MD-RS) were established, to emphasize the importance of identifying forms with a potential therapeutic target [Arber 2016].Luspatercept is a recombinant fusion protein consisting of the modified extracellular domain of human activin receptor type IIB (ActRIIB) linked to a domain of human immunoglobulin Fc G1. The drug showed efficacy in re-establishing erythropoiesis and was recently approved for the treatment of adult patients with very low- to intermediate-risk MDS-RS failing ESAs[Platzbecker 2017; Fenaux 2020].AimsTo retrospectively assess the efficacy and/or clinical benefit (defined as a reduction in red blood cells transfusions [RBCT] or increase in hemoglobin levels) of luspatercept in patients with LR MDS with RS in a real-life setting. Safety issues were also addressed and clinical and hematological predictors of outcome analyzed.MethodsWe considered all IPSS-R lower-risk MDS with RS patients with unsatisfactory response or unsuitable for ESAs, treated with luspatercept as monotherapy through an expanded access program since November 2020, in 2 tertiary hematologic centers in Milan, Italy. Luspatercept was administered according to the published schedule: starting dose 1,0 mg/kg every three weeks, with the possibility to increase up to 1,33 mg/kg and 1,75 mg/kg if patients did not reach transfusion independence or decreased transfusion burden after two consecutive infusions. Response to luspatercept was evaluated according to IWG 2018 criteria: Hb increase by > 1.5 g/dL and/or relevant reduction of units of RBC transfusions by an absolute number of at least 4 RBC transfusions/8 wk compared with the pretreatment transfusion number in the previous 8 wk.ResultsWe included a total of 17 pts (70% males, 30% females), with a median age of 68 years (range 45-89). The median follow-up from the start of the Luspatercept was 4 months (range 3-6).According to WHO 2017 we included 13 MLD (76%) and 4 ULD (24%); in all of them SF3B1 mutations was confirmed. Moreover, According to IPSS-R classification: 5 (27%)cases were intermediate and 12 (73%) low risk.All patients received at least three doses of luspatercept. Nine patients (53%) needed a dose escalation to 1,33 mg/kg, and 7 (41%) required the maximal dose of 1,75 mg/kg. Interestingly, not all patients received a dose escalation, and 8 (47%) obtained prolonged efficacy with 1 mg/kg dose.Hemoglobin levels increased in 9 pts (53%): 4 (24%), 3 (18%) and 2 (12%) cases at 1 mg/Kg, 1,33 mg/kg and 1,75 mg/dl, respectively. Medina increase was 1,5 g/dl (range 0,2 - 2,2) and these levels were maintained for a median of 4 months (range 3-6).In addition, considering transfusion burden, the independence was reached in 3 pts (18%), after a median time of 6 weeks from treatment start. Importantly, off the 14 patients remaining transfusion-dependent, 6 (35%) reduced the transfusion burden.The most prominent toxicity was increased blood pressure (grade 2) occurring in 3 pts (18%), and dizziness (grade 2) in 2 pts (12%); though the drug was generally well tolerated. No serious adverse events (SAEs) were reported. Therefore, patients were treated as outpatients in all cases.At the last follow up, all patients are alive and free from leukemic evolution.Conclusions:Our real-world experience confirms the efficacy of luspatercept in improving erythropoiesis and decreasing transfusion dependency in very low, low or intermediate risk MDS with RS, with an acceptable toxicity profile. DisclosuresFattizzo: Amgen: Honoraria, Speakers Bureau; Apellis: Speakers Bureau; Annexon: Consultancy; Momenta: Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Alexion: Speakers Bureau; Kira: Speakers Bureau. Barcellini: Agios: Honoraria, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals: Honoraria; Novartis: Honoraria.

The Genetics of Myelodysplastic Syndromes: Clinical Relevance.

Myelodysplastic syndromes (MDS) are a clonal disease arising from hematopoietic stem cells, that are characterized by ineffective hematopoiesis (leading to peripheral blood cytopenia) and by an increased risk of evolution into acute myeloid leukemia. MDS are driven by a complex combination of genetic mutations that results in heterogeneous clinical phenotype and outcome. Genetic studies have enabled the identification of a set of recurrently mutated genes which are central to the pathogenesis of MDS and can be organized into a limited number of cellular pathways, including RNA splicing (SF3B1, SRSF2, ZRSR2, U2AF1 genes), DNA methylation (TET2, DNMT3A, IDH1/2), transcription regulation (RUNX1), signal transduction (CBL, RAS), DNA repair (TP53), chromatin modification (ASXL1, EZH2), and cohesin complex (STAG2). Few genes are consistently mutated in >10% of patients, whereas a long tail of 40–50 genes are mutated in <5% of cases. At diagnosis, the majority of MDS patients have 2–4 driver mutations and hundreds of background mutations. Reliable genotype/phenotype relationships were described in MDS: SF3B1 mutations are associated with the presence of ring sideroblasts and more recent studies indicate that other splicing mutations (SRSF2, U2AF1) may identify distinct disease categories with specific hematological features. Moreover, gene mutations have been shown to influence the probability of survival and risk of disease progression and mutational status may add significant information to currently available prognostic tools. For instance, SF3B1 mutations are predictors of favourable prognosis, while driver mutations of other genes (such as ASXL1, SRSF2, RUNX1, TP53) are associated with a reduced probability of survival and increased risk of disease progression. In this article, we review the most recent advances in our understanding of the genetic basis of myelodysplastic syndromes and discuss its clinical relevance.

Somatic PRPF8 Mutations in Myeloid Neoplasia

Somaticmutations in genes of the RNA-splicing machinery are recurrent in myeloid neoplasia. PRPF8 has a central role in the spliceosome assembly and it is required in all tissues. Somatic mutations in PRPF8 have been described in patients with MDS. We previously showed that PRPF8 mutations (PRPF8MT) are associated with presence of ring sideroblasts (RS) and a distinct myeloid phenotype. However, to date, no comprehensive analysis has been conducted to unravel the clonal architecture of PRPF8MTpatients.We took advantage of a large database of patients with bone marrow failure syndromes (BMFS) annotated for molecular and clinical characteristics (n=1700; median follow up 1.0 year, range 1-5 years), median age was 71 years (range, 13-93). Whole exome sequencing and targeted deep sequencing of a panel of 65 genes was applied. Our analyses included somatic mutational patterns, clonal hierarchy, and cross-sectional correlation in the cohort of PRPF8MT and PRPF8 wild type.PRPF8 mutations were found in 4% (65/1700) of patients. The mutations were mainly missense but nonsense, frameshift, and splice mutations were also seen. No specific hotspot was identified, although we noted that mutations at D1958 were detected in 7 patients, F1099 in 4 patients and C1594 and V1015 mutations were detected in 3 patients each. 6 mutations (-17/17p-) were consistent with hemizygous configuration of PRPF8MT.A total of 368 mutations in the 65 gene panel were detected. Median number of mutations/ patient was 3 (range 1-26). PRPF8MT cases had a median overall survival (OS) of 26 months (mo) (range 8-110). 48/65 samples were tested for the presence of RS, showing 31% of samples with RS (RS+) and 69% of samples without RS (RS-). PRPF8MT/RS+ were mainly MDS (47%) and MDS/MPN (27%) while PRPF8MT/RS- were distributed in MDS (27%), BMFS (AA, PRCA, PNH; 27%), and AML (24%). We found that PRPF8MT/RS+ had a higher neutrophil counts vs. PRPF8MT/RS- (3.2±2.2 vs. 1.7±0.02) as well as platelets counts (158±197 vs. 91±150; P=.06) and a mild decreased number of lymphocytes (1.0±0.6 vs. 1.8±2.3, P=.03) perhaps due to a higher proportion of patients with BMFS. PRPF8MT were mostly normal karyotype (45%) with a low percentage of complex karyotypes (16%)We dissected the clonal hierarchy of PRPF8MT and found that 38% (25/65) were ancestral while 62% (40/65) were secondary hits. The most commonly ancestral mutations besides PRPF8 were TET2 (n=3) and SF3B1 (n=3). The most common secondary mutations after PRPF8 were TET2 (n=14) and ASXL1 (n=14). In MDS and AML, PRPF8MT were either ancestral or secondary, while in MDS/MPN and BMFS, PRPF8MT was mostly a secondary mutation (71% and 75%, P=.05 and P=.04).We further analyzed the mutational spectrum of PRPF8MT patients. Cross-sectional analysis identified that the top mutated genes in PRPF8MT clustered in epigenetic regulation and histone modification (19%; TET2, DNMT3A, IDH1/2, EZH2, ASXL1, KDM6A), followed by transcriptional regulation (13%; RUNX1, BCOR/L1, PHF6, ETV6, TP53), RNA-splicing (12%; SRSF2, SF3B1, LUC7L2, U2AF1, ZRSR2, DDX41, DHX29), and DNA-repair (7%; CUX1). Unlike other splicing mutations PRPF8 coexisted with other splicing lesions: SF3B1 (12%), SRSF2 (6%), LUC7L2 (9%), U2AF1 (8%), and ZRSR2 (6%). Of note, we found rare cases with triple splicing factor mutations (n=6). When we assessed the impact of the clonal architecture, ancestral PRPF8MT had a shorter OS than those with secondary mutations (35mo vs. 59mo; P=.04).In sum,clonal architecture analyses showed that PRPF8MT are frequently secondary rather than ancestral mutations but when are ancestral hits, they lead to dismal prognosis. Unlike the more common spliceosomal factor mutations (SF3B1, U2AF1, SRSF2) that rarely co-occur, PRPF8MT often co-occur with other spliceosomal mutations. This may be due to the fact that PRPF8MT are mostly secondary mutations. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) and MDS/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T) - "2021 update on diagnosis, risk-stratification, and management".

Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include myelodysplastic syndromes with RS (MDS-RS) and MDS/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). MDS-RS is a lower risk MDS, with single or multilineage dysplasia (MDS-RS-SLD/MLD), <5% bone marrow (BM) blasts, <1% peripheral blood blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥450 × 109 /L and large atypical megakaryocytes. Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F (50%), DNMT3A, TET2 and ASXL1 mutations. Cytogenetic abnormalities are uncommon in both. Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-IPSS. Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia (MPN). Both diseases are associated with a low risk of leukemic transformation. Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Luspatercept, a first-in-class erythroid maturation agent is now approved for the management of anemia in patients with MDS-RS and MDS/MPN-RS-T. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs remains to be defined.

SF3B1 mutant myelodysplastic syndrome: Recent advances

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes encoding different components of the spliceosome occur in more than half of all MDS patients. SF3B1 is the most frequently mutated splicing factor gene in MDS, and there is a strong association between SF3B1 mutations and the presence of ring sideroblasts in the bone marrow of MDS patients. It has been recently proposed that SF3B1 mutant MDS should be recognized as a distinct nosologic entity. Splicing factor mutations cause aberrant pre-mRNA splicing of many target genes, some of which have been shown to impact on hematopoiesis in functional studies. Emerging data show that some of the downstream effects of different mutated splicing factors converge on common cellular processes, such as hyperactivation of NF-κB signaling and increased R-loops. The aberrantly spliced target genes and the dysregulated pathways and cellular processes associated with splicing factor mutations provided the rationale for new potential therapeutic approaches to target MDS cells with mutations of SF3B1 and other splicing factors.

Toxic iron species in lower-risk myelodysplastic syndrome patients: course of disease and effects on outcome

Toxic iron species in lower-risk myelodysplastic syndrome patients: course of disease and effects on outcome

Novel frameshift variant (c.409dupG) in SLC25A38 is a common cause of congenital sideroblastic anaemia in the Indian subcontinent

AimsCongenital sideroblastic anaemias (CSAs) are a group of rare disorders with the presence of ring sideroblasts in the bone marrow. Pathogenic variants are inherited in an autosomal recessive/X-linked fashion. The...

SF3B1 Mutation but Not Ring Sideroblasts Identifies a Specific Group of Myelodysplastic Syndrome–Refractory Cytopenia With Multilineage Dysplasia

BackgroundIntegrating the proportion of ring sideroblasts and SF3B1 mutation status is required for diagnosis of sideroblastic subgroups in myelodysplastic syndrome (MDS) as proposed by the World Health Organization 2016 classification. However, the clinical implications of SF3B1 mutation and ring sideroblasts in MDS-refractory cytopenia with multilineage dysplasia (MDS-RCMD) remain unclear. Patients and MethodsClinical and laboratory features in 238 MDS-RCMD patients were retrospectively analyzed, and the prognostic significance of SF3B1 mutation and ring sideroblasts on overall survival and leukemia-free survival in total MDS-RCMD patients and different subgroups stratified by the percentage of ring sideroblasts or SF3B1 mutation status were evaluated. ResultsMDS-RCMD patients with ring sideroblasts ≥ 15% showed a significantly higher prevalence of SF3B1 mutation compared to ring sideroblasts 5%-14% or ring sideroblasts < 5% (75.6% vs. 15.1% vs. 6.4%, P < .001). In multivariate analysis, SF3B1 mutation was associated with a significantly prolonged survival (hazard ratio [HR] = 0.430, P = .013) and reduced leukemic transformation (HR = 0.174, P = .021) in total MDS-RCMD patients, while ring sideroblasts showed no independent effect on either survival or leukemic transformation. There were no significant differences in clinical characteristics or survival between MDS-RCMD patients with ring sideroblasts ≥ 15% and ring sideroblasts 5%-14% in the presence of SF3B1 mutation. Furthermore, SF3B1 mutation showed an independent prognostic effect on overall survival in MDS-RCMD patients with ring sideroblasts 5%-14% (HR = 0.195, P = .046). ConclusionSF3B1 mutation, not the presence of ring sideroblasts, identifies a distinct subtype and showed independent prognostic value on survival and leukemia transformation in MDS-RCMD patients.

Molecular Characterization and Novel Therapeutic Strategy for X-Linked Sideroblastic Anemia Associated with ALAS2 Missense Variants

Background: Congenital sideroblastic anemia (CSA) is an inherited anemia characterized by the presence of ring sideroblasts (RSs) in bone marrow. X-linked sideroblastic anemia (XLSA) is the most common form of CSA and is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Each ALAS2 mutation has a variable effect on the protein's enzymatic function, and only half of XLSA patients are responsive to pyridoxal 5′-phosphate (PLP) treatment. Thus, novel therapeutic strategies should be explored. We have established an in vitro XLSA model in human induced pluripotent stem cell-derived erythroid progenitors (HiDEP-1) by disrupting GATA-1-binding intronic enhancer region of ALAS2, which accounts for a minor XLSA subtype with PLP refractoriness (Saito and Ono et al. MCB 2019; ASH 2018). However, evidence regarding molecular characterization of ALAS2 missense variants is lacking, presumably due to difficulties in introducing the desired missense mutations in primary erythroblasts. In this study, we optimized a protocol for introducing point mutations in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells and subsequently characterized XLSA models harboring ALAS2 missense variants. Methods: Based on homology-directed CRISPR/Cas9 system, we introduced an ALAS2 mutation from arginine at amino acid residue 170, one of the XLSA hot spots, into leucine (R170L) or histidine (R170H). A twenty-nucleotide targeted genomic sequence, located upstream of protospacer adjacent motif (PAM) near ALAS2 R170, was cloned into plasmid vectors expressing Cas9 nuclease and green fluorescent protein (GFP). Each homologous recombinant template was designed as a 127 single-stranded oligodeoxynucleotide (ssODN) asymmetrically distributed from the Cas9-mediated cleavage site. PAM silent mutations were added to avoid continuous cleavage after successful mutation. HUDEP-2 cells were co-transduced with gRNA/Cas9/GFP-expressing vectors and ssODNs using Amaxa Nucleofector 2b (Lonza). After 48 hours, cells were isolated by GFP-positive expression; subsequently, single-cell dilution was performed, followed by clonal analysis. Results: Both XLSA clones (ALAS2 R170L and R170H) appeared pink/pale colored, reflecting impaired hemoglobin biosynthesis. Consistently, quantitative RT-PCR analysis demonstrated decreased globin gene (HBB) expression. On the other hand, XLSA clones did not show RSs and were morphologically similar to wild-type controls. When XLSA clones were induced to undergo erythroid differentiation by co-culturing with OP9 cells in a 100 mM sodium ferrous citrate (SFC)-supplemented medium, increased numbers of RSs were observed in mutant clones, mimicking findings in XLSA patients. Electron microscopy confirmed an aberrant mitochondrial iron deposit in XLSA clones. Also, HBB expression and intracellular heme concentration were significantly lower than those in wild-type controls. Intriguingly, while OP9 co-culture and SFC addition promoted erythroid differentiation in both wild-type and XLSA clones, XLSA clones exhibited a more immature morphological phenotype than wild-type controls. Expression profiling revealed that 317 and 86 genes were commonly up- and downregulated &amp;gt;2-fold in XLSA clones compared with those in wild-type controls. Gene ontology analysis showed significant (p &amp;lt; 0.01) enrichment of genes associated with mitochondrial gene expression and organization, suggesting that mitochondrial alteration is involved in XLSA pathogenesis. On the other hand, 188 genes in total were differentially expressed between ALAS2 R170L and R170H. Genes associated with ferroptosis, such as GPX4 and GCLC, showed a significantly lower expression in ALAS2 R170H clones, possibly accounting for phenotypic differences among patients with ALAS2 R170L or R170H mutation. Finally, supplementation with 5-aminolevulinic acid (ALA) significantly improved the compromised heme biosynthesis in XLSA clones, suggesting ALA treatment to be a promising therapeutic option for XLSA. Conclusion: The established models may act as useful tools for exploring the precise molecular mechanisms of XLSA harboring missense mutations and for drug testing. Also, our homology-directed CRISPR/Cas9-based protocol can be applied to establish a wide variety of CSA as well as congenital anemia models. Disclosures Fukuhara: Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Gilead: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Celgene Corporation: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; AbbVie: Research Funding; Bayer: Research Funding; Solasia Pharma: Research Funding. Onishi:Celgene: Honoraria; Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria.

Diagnostic Testing Patterns and Concordance with World Health Organization (WHO) Criteria for Patients (Pts) with Newly Diagnosed (ND) Myelodysplastic Syndromes (MDS) in the Connect® MDS/AML Registry

Diagnostic Testing Patterns and Concordance with World Health Organization (WHO) Criteria for Patients (Pts) with Newly Diagnosed (ND) Myelodysplastic Syndromes (MDS) in the Connect® MDS/AML Registry

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.

Impact of treatment with iron chelation therapy in patients with lower-risk myelodysplastic syndromes participating in the European MDS registry.

Iron overload due to red blood cell (RBC) transfusions is associated with morbidity and mortality in lower-risk myelodysplastic syndrome (MDS) patients. Many studies have suggested improved survival after iron chelation therapy (ICT), but valid data are limited. The aim of this study was to assess the effect of ICT on overall survival and hematologic improvement in lower-risk MDS patients in the European MDS registry. We compared chelated patients with a contemporary, non-chelated control group within the European MDS registry, that met the eligibility criteria for starting iron chelation. A Cox proportional hazards model was used to assess overall survival (OS), treating receipt of chelation as a time-varying variable. Additionally, chelated and non-chelated patients were compared using a propensity-score matched model. Of 2,200 patients, 224 received iron chelation. The hazard ratio and 95% confidence interval for OS for chelated patients, adjusted for age, sex, comorbidity, performance status, cumulative RBC transfusions, Revised-International Prognostic Scoring System (IPSS-R), and presence of ringed sideroblasts was 0.50 (0.34-0.74). The propensity-score analysis, matched for age, sex, country, RBC transfusion intensity, ferritin level, comorbidity, performance status, and IPSS-R, and, in addition, corrected for cumulative RBC transfusions and presence of ringed sideroblasts, demonstrated a significantly improved OS for chelated patients with a hazard ratio of 0.42 (0.27-0.63) compared to non-chelated patients. Up to 39% of chelated patients reached an erythroid response. In conclusion, our results suggest that iron chelation may improve OS and hematopoiesis in transfused lower-risk MDS patients. This trial was registered at clinicaltrials.gov identifier: 00600860.

S838 TRANSFUSION DEPENDENCY IS ASSOCIATED WITH PRESENCE OF TOXIC IRON SPECIES AND INFERIOR SURVIVAL IN PATIENTS WITH LOWER-RISK MYELODYSPLASTIC SYNDROMES

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.

S837 TREATMENT WITH IMETELSTAT PROVIDES DURABLE TRANSFUSION INDEPENDENCE (TI) IN HEAVILY TRANSFUSED NON‐DEL(5Q) LOWER RISK MDS (LR‐MDS) RELAPSED/REFRACTORY (R/R) TO ERYTHROPOIESIS STIMULATING AGENTS (ESAS)

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 &gt;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 &gt;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.

EPO-R+ myelodysplastic cells with ring sideroblasts produce high erythroferrone levels to reduce hepcidin expression in hepatic cells

Recently, a new erythroid regulator, erythroferrone (ERFE), which downregulates hepatic hepcidin production, has been identified. However, the relationship between ERFE and abnormal iron metabolism in MDS is unclear. In this study, we examined the level of ERFE mRNA during ex vivo erythroid differentiation using cord blood CD34+ cells and we further analyzed whether ERFE could be produced by MDS cells using a public database (GSE58831). ERFE mRNA was increased during normal erythroid differentiation. An analysis of GSE58831 indicated that ERFE expression in bone marrow (BM) MDS cells was higher than that in healthy volunteer (HV)–derived BM cells. ERFE expression significantly and positively correlated with the expression of erythropoietin (EPO) receptors (EPO-R), ALAS2 (5’-Aminolevulinate Synthase 2), STEAP3 (STEAP family member 3) and the presence of ring sideroblasts or the SF3B1 mutation. These results suggest that EPO-R+ MDS cells with ring sideroblasts or an SF3B1 mutation produce high levels of ERFE that may be associated with a reduction in hepcidin.