Refractory Anemia With Ringed Sideroblasts Research Articles

GDF11/SMAD Regulated Splicing of GATA1 Is Associated with Response to Luspatercept in Lower-Risk Myelodysplastic Syndromes (LR-MDS)

Introduction: Luspatercept is an erythroid maturation agent that promotes late-stage erythropoiesis in patients with LR-MDS. While it is known that luspatercept, a modified activin receptor ligand trap, binds to GDF11 and Activin B, the mechanism by which these ligands trigger a decrease in red blood cell (RBC) formation is unknown. Luspatercept was approved by the FDA following the phase 3 MEDALIST study, but biomarkers of response are currently unknown. Results:In a large cohort of primary MDS CD34+ samples, GDF11 expression was significantly higher in MDS samples than in age-matched healthy controls (183 MDS samples vs 17 controls, P < 0.05). GDF11 was detected in primary MDS sera and was substantially elevated in the refractory anemia (RA) and RA with ring sideroblasts (RARS) MDS subgroups. Activin receptor 2B (ACVR2B), the receptor of GDF11, was also overexpressed in MDS samples, and its expression was confirmed by immunohistochemistry (IHC) in the bone marrows of primary MDS patients. GDF11/ACVR2B activates SMAD2, and we found that the overexpression of SMAD2 in CD34+ cells was associated with more severe anemia in MDS patients. RNA-seq and SMAD2 ChIP-seq analyses were then performed to determine the downstream effects of GDF11 in erythroid progenitors. Human CD34+ primary cells were utilized to generate BFU-E stage erythroid progenitors. We discovered that GDF11 promotes an abnormal erythroid gene expression program characterized by increased expression of pro-apoptotic and early erythroid differentiation-related genes. SMAD2 binding was detected in regulatory regions of early erythroid (including GATA1), proinflammatory, and apoptotic transcripts using ChIP-seq analysis with p-SMAD2. In a zebrafish model, administration of GDF11 caused a decrease in erythroid cells, which was rescued by treatment with luspatercept (Wobus et al. Leukemia 2021 35(10):2936-47). Luspatercept inhibited the phosphorylation and activation of SMAD2 in response to GDF11 stimulation in primary hematopoietic cells and leukemia cell lines. Importantly, luspatercept could reverse the growth-inhibiting effects of MDS-derived sera on primary hematopoietic progenitors. Finally, to identify biomarkers associated with clinical responses, we analyzed phase 3 MEDALIST study samples collected before and after treatment with lupatercept. RNA-seq analysis was performed on bone marrow samples from responders (R; n = 14) and non-responders (NR; n = 9) (Figure). Analysis of pre-treatment/screening bone marrow transcriptomes revealed no significant differences between R and NR in global gene expression. Analysis of splice isoforms between the two cohorts revealed strikingly greater differences. Most differential splicing events were observed in NR (702 in R vs 5232 in NR). The most prevalent splicing modifications were exon skipping events, and most functional pathways affected by differential exon usage were RNA splicing related. GATA1, the master erythroid transcription factor, was spliced differently in R at baseline, which was an intriguing observation. Response to luspatercept was found to be associated with exon 2 skipping, resulting in a shortened isoform of GATA1. R had a greater proportion of the short GATA1 isoform than NR (12% in R versus 6% in NR; P = 0.04). We analyzed ChIP-seq data from primary erythroid cells to determine the effect of GDF11/SMAD2 activation on GATA1 splicing. GDF11 stimulation resulted in SMAD2 binding to the second intron of GATA1. CRISPR-mediated deletion of the SMAD2 binding region in primary erythroid progenitors increased expression of the GATA1 long isoform. Next, we performed a CRISPR-mediated deletion of exon 2 of GATA1, resulting in a predominance of GATA1 short isoform expression and a decrease in erythroid output. In CRISPR-modified primary erythroid cells with a predominant short GATA1 isoform (exon 2-deleted cells), luspatercept was able to reverse the inhibition induced by GDF11. Conclusions: These results demonstrate that MDS cases that respond to luspatercept are associated with a relatively high level of expression of functionally deficient GATA1 short isoform. We further demonstrate that GDF11 stimulates the production of the GATA1 short isoform via SMAD2 binding to intron 2 of the GATA1 gene. The regulation of a GATA1 splicing isoform by GDF11/SMAD2 is associated with clinical responses to luspatercept, according to these novel findings.

De Novo Germline DHX38 Variant Associated with Alternative Splicing of Multiple Transcripts in Iron-Related Pathways in a Patient with Atypical Congenital Dyserythropoietic Anemia with Ring Sideroblasts

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare hereditary anemias caused by ineffective and dyspoietic erythropoiesis, with the pathognomonic feature of binuclear and/or multinuclear erythroid precursors in the bone marrow. The clinical diagnosis of CDA is based on identifying the characteristic morphology of erythroblasts in the bone marrow of patients presenting with chronic anemia, frequently with evidence of hemolysis but suboptimal reticulocytosis, and iron overload. CDAR (ClinicalTrials.gov identifier: NCT02964494), a registry for patients with CDA in North America, has been established with the goal of providing a longitudinal database and associated biorepository to facilitate natural history studies and research on the pathogenetic mechanisms in this heterogenous disease. A 2-year-old female, enrolled in CDAR, had transfusion-dependent anemia since birth. She demonstrated accelerated iron overload and had failure to thrive despite regular transfusions maintaining trough hemoglobin higher than 9.5 g/dL. Bone marrow evaluation revealed dyserythropoiesis along with ring sideroblasts (RS), resembling acquired myelodysplastic syndrome (MDS) with the characteristics of Refractory Anemia with RS (RARS). Whole-exome sequencing for the proband and her parents in trio analysis identified a de novo, heterozygous germline DHX38 missense variant c.3310A>C, p.Thr1104Pro in the proband. In silico prediction programs suggest this rare variant is deleterious. DEAH box RNA helicase DHX38 is an RNA helicase, homolog to the yeast Prp16 that has been shown to play a dual role in the spliceosome complex: an ATP-independent role in the first catalytic step mediating proofreading of the branchpoint sequence, and an ATP-dependent role in the second step, mediating removal of the lariat and joining the exons to complete the splicing (Tseng et al, RNA 2011). DHX38 is highly expressed during terminal erythropoiesis similarly to SF3B1, the splicing factor gene most frequently mutated in MDS-RARS. RNA-seq analysis of CD71+ reticulocytes isolated from the proband at three different time points compared to three different normal control samples, using AltAnalyze software, revealed approximately 3,000 differentially expressed genes. More than 1,500 differentially spliced events were identified, affecting over 200 genes, including SLC25A37, coding for an iron importer localized in the mitochondrial inner membrane and PPOX, coding for protoporphyrinogen oxidase (Figure 1A), both essential for the synthesis of mitochondrial heme. Mis-splicing of the same genes is a characteristic finding in MDS-RARS associated with SF3B1 pathogenic variants, most commonly p.Lys700Glu, and may contribute to RS formation. Moreover, DHX38 is known to interact with centromeric noncoding RNA, regulating chromosome segregation during mitosis, and its depletion in vitro causes abnormal grape-shaped nuclear morphology (Nishimura et al, Genes Cells 2019). Review of the patient's bone marrow studies revealed several erythroblasts with grape-shaped nuclei (Figure 1B). Erythropoiesis cultures from patient-derived iPSCs were utilized to phenocopy the disease in vitro. Cytospins of the generated erythroblasts revealed dyserythropoiesis that was verified quantitatively by increased frequency of nuclear abnormalities in erythroblasts produced in vitro from patient-derived iPSCs analyzed by imaging flow cytometry. Extra-hematopoietic abnormalities of the nucleus and mitotic defects were explored in fibroblasts generated from the patient's skin biopsy, which also tested positive for DHX38 p.Thr1104Pro. Grape-shaped nuclei were detected in cultures of patient's fibroblasts indicating improper mitotic exit due to aberrant chromosome segregation. Our findings indicate that DHX38 is likely the causative gene for this CDA with a RARS phenotype, causing mis-splicing of a multitude of genes critical for iron metabolism and erythropoiesis as well as defects in chromosome segregation and cell division. Out of approximately 250,000 alleles in the gnomAD database, the DHX38 p.Thr1104Pro variant has been detected in 4 alleles; however, SF3B1 p.Lys700Glu is also detected in 22. Therefore, the rare presence of this DHX38 variant in "healthy" population may indicate that acquired DHX38 pathogenic variants exist and could be responsible for SF3B1-negative MDS-RARS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Incidence and survival estimates for patients with myelodysplastic syndrome in the early 21st century: no evidence of improvement over time

We examine changes in population level incidence and survival of patients diagnosed with myelodysplastic syndrome (MDS) in the United States in 2001–2016. Data were extracted from the Surveillance, Epidemiology, and End Results (SEER)-18 database. Period analysis was used to calculate one-, two-, and five-year survival. The incidence peaked at 5.6 per 100,000 in 2010 then decreased to 3.9 by 2016, with a decrease in the diagnoses of refractory anemia (RA) and RA with ringed sideroblasts (RARS) and a relative increase in RA with excess blasts (RAEB). Overall, one-, two-, and five-year relative survival decreased over time, going from 74.3%, 60.9%, and 42.3%, respectively, in 2008–2010 to 70.9%, 55.9%, and 37.6%, respectively, in 2014–2016. When survival was examined by histology, patients with RA/RARS and RAEB had similar survival expectations in 2008–2010 versus 2014–2016 and a decrease was observed for 5q-MDS. Our results highlight the need for new treatment options in MDS.

Clinical Application for Diagnosis of Myelodysplatic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis.

Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/ MPN-RS-T) was newly introduced as a full entity in the 2016 revision of the WHO classification. In this study, we investigated the morphologic, laboratory, and clinical features of MDS/MPN-RS-T. We reviewed the bone marrow and genetic studies of patients whose diagnoses were coded as "refractory anemia with ring sideroblasts (RARS)" or "MDS/MPN, unclassifiable" between January 2008 and April 2018. A total of 8 cases fulfilled the criteria for a diagnosis of MDS/MPN-RS-T. All of them had no specific symptoms. Half of the cases had less than 450 × 109/L platelet counts by an automated hematology analyzer; however, all platelet counts exceeded 450 × 109/L when performed manually. JAK2 mutation tests were performed in 7 cases, and a heterozygous mutation was detected in 1 case. SF3B1 mutations were present in 3 of the 4 cases tested. When RARS is suspected in patients without thrombocytopenia, manual platelet counts should be performed. For patients with suspected essential thrombocythemia, RS evaluation through careful observation of an iron-stained slide is crucial. Since the independent evaluation of RS was reflected in the revised classification, the ambiguous disease classification becomes clearer and more consistent.

Clinical effectiveness of DNA methyltransferase inhibitors and lenalidomide in older patients with refractory anemia with ring sideroblasts: a population-based study in the United States

Existing studies regarding the role of DNA methyltransferase inhibitors (DNMTi) and lenalidomide in refractory anemia with ring sideroblasts (RARS) are limited. Using the surveillance, epidemiology, and end results-medicare database, we assembled a population-based cohort of older adults diagnosed with non-del(5q) lower-risk myelodysplastic syndromes during 2007–2015. Of 2167 patients, 30% had RARS. About 16% of RARS and non- ring sideroblasts (RS) patients received DNMTi. RARS patients were more likely to receive lenalidomide (11.1% vs. 7.1%, p < 0.01). Among patients who were transfusion-dependent at treatment initiation, 55.6% of those treated with DNMTi only and 42.5% treated with lenalidomide only achieved red blood cell transfusion independence (RBC-TI) for a median duration of 21 and 12 weeks, respectively. RS status did not impact rate of RBC-TI. RARS patients had a significantly better survival, and the median survival of RARS patients varied by treatment group. In this population-based study of older RARS patients, DNMTi and lenalidomide were clinically active.

Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes

Early erythroblasts from patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) show constitutive mitochondrial release of cytochrome c. Moreover, mature erythroblasts in RARS, but not in RA, display aberrant accumulation of mitochondrial ferritin (MtF). We analyzed cytochrome c release, MtF expression, and gene expression during erythroid differentiation in bone marrow cells from myelodysplastic syndrome (MDS) patients and healthy controls. Whereas none or few cultured erythroid cells from healthy individuals and RA patients expressed MtF, those from RARS patients showed MtF expression at an early stage, when cells were CD34+ and without morphologic signs of erythroid differentiation. The proportion of RARS erythroblasts that were MtF+ increased further upon in vitro maturation. Moreover, a significant overexpression of mRNA encoding cytochrome c, and proapoptotic Bid and Bax, was seen in freshly isolated cells from MDS patients. Genes involved in erythroid differentiation were also dysregulated in MDS cells. Importantly, GATA-1 expression increased during normal erythroid maturation, but remained low in MDS cultures, indicating a block of erythroid maturation at the transcriptional level. In conclusion, aberrant MtF expression in RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in this process.

Clinical Effectiveness of Hypomethylating Agents (HMAs) and Lenalidomide (Len) in Older Patients (pts) with Refractory Anemia with Ring Sideroblasts: A Large Population-Based Study in the United States (US)

Clinical Effectiveness of Hypomethylating Agents (HMAs) and Lenalidomide (Len) in Older Patients (pts) with Refractory Anemia with Ring Sideroblasts: A Large Population-Based Study in the United States (US)

Current status of decitabine in refractory anemia with ringed sideroblasts

Refractory anemia with ringed sideroblasts (RAS) is one of myelodysplastic syndrome (MDS) in the French-American-British cooperative group (FAB) classifications, and it often belongs to a relatively low-risk group of MDS. The main therapeutic goals for the usage of demethylated drugs for RAS patients are improving cytopenia, detaching from blood transfusion, and improving quality of life and prolonging survival. Decitabine (DAC) is an important demethylating drug that plays an important role in the treatment of RAS. Currently, the optimal therapeutic dose for DAC treatment of RAS is 20 mg/(m2·d). Since larger dose of DAC may cause more severe levels of myelosuppression, recently DAC has shown a decreasing trend in therapeutic dose for lower-risk MDS, which including RAS. This article summarizes the current status in pathogenesis of RAS, and the mechanisms, optimal dosage and adverse reactions of DAC treatment in RAS, in order to provide theoretical basis for clinical treatment of RAS. Key words: Myelodysplastic syndromes; Anemia, refractory; Anemia, sideroblastic; DNA methylation; Decitabine

Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis: "2019 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 refractory anemia with ring sideroblasts (RARS), now classified under myelodysplastic syndromes with RS (MDS-RS) and RARS with thrombocytosis (RARS-T); now called myelodysplastic/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). MDS-RS is a lower-risk MDS, with single or multilineage dysplasia (SLD/MLD), <5% bone marrow (BM) 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 × 10(9)/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, ASXL1, DNMT3A, SETBP1, and TET2 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. Both diseases have 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. The advent of luspatercept, a first-in-class erythroid maturation agent will tremendously boost the ability to manage anemia. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs remains uncertain.

Lenalidomide Is Effective Treatment Option for Patients with Refractory Anemia with Ring Sideroblasts and Thrombocytosis

CONTEXT AND OBJECTIVE:Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) is a provisional entity included in the MDS/MPN overlap syndromes, with features of refractory anemia with ring sideroblasts (RARS) and thrombocytosis (platelet count ³ 450 x 10/L), recently renamed MDS/MPN with ring sideroblasts and thrombocytosis. Management of RARS-T is extrapolated from MDS and MPN treatment options namely for alleviating symptoms of anemia and, occasionally, for thrombocytosis. Individual case reports have suggested that lenalidomide may be effective in treating RARS-T. In non-del 5q, low-risk MDS, 25% of patients (pts) became red blood cell transfusion independent (RBC-TI). In the phase II study of lenalidomide in non-del 5q lower risk MDS, 35% of pts with RARS treated with lenalidomide achieved RBC-TI. The primary objective of this study is to evaluate a series of pts with RARS-T and compare hematological improvement (HI) responses among different treatment options.METHODS:This is a retrospective, single-center study. Patients with RARS-T were identified from the MDS Database at Moffitt Cancer Center. Samples for somatic mutation testing were obtained at time of referral. Descriptive statistics were used to describe baseline characteristics and response rates to treatment. The primary endpoint was HI using international working group criteria (IWG) 2006. The median overall survival (OS) was calculated from time of diagnosis.RESULTS:We identified 33 pts with RARS-T. The mean age was 67 years (38-80), 15 pts were male (45.5%), and majority were Caucasians (n=27, 82%). According to international prognostic scoring system (IPSS), 24 patients (73%) were low risk and 9 pts (27%) were int-1 risk. By revised IPSS, 11 pts (33%), 18 (55%), 3 (9%), and 1 (3%) were very low, low, intermediate, and high risk, respectively. The mean WBC was 7.7 (2.8-25.7), the mean Hemoglobin was 9.9 g/dl (7.3-12.4), and the mean platelet count was 682 (372-1644). Among 20 pts who had Next generation sequencing for somatic mutations at time of referral, 19 pts (95%) had SF3B1 mutation and 4 pts (20%) had JAK-2 V617 F mutation.The median OS was 90 mo (95% CI 47-134). For pts with JAK-2 V617F mutations, the median OS was 110 mo versus 74 mo for those with wild type (p=0.24). The rate of AML transformation was 3% (one patient only).Among the 33 pts, 8 pts had no treatment (observation only), 22 pts (67%) received erythroid stimulating agent (ESA) prior to any active therapy, 2 patients received lenalidomide with no prior ESA, and 1 pt ruxolitinib with no prior ESA. Twelve pts (36%) received lenalidomide (7 as first line active therapy after ESA and 2 without prior ESA exposure) and 13 pts (33%) received azacitidine (6 as first line active therapy after ESA).The HI rate for ESA was 36% (12/22) with a median duration of ESA treatment of 33mo. The HI rate for lenalidomide was 50% (6/12) with a median duration of lenalidomide treatment of 10.3mo. Among the 9 pts who received lenalidomide as first line active therapy (2 pts without prior ESA exposure) HI rate was 56%. The HI rate for azacitidine was 15% (2/13) with a median duration of azacitidine treatment of 6.3mo.CONCLUSIONS: Pts with RARS-T had favorable outcomes, with long overall survival and a low rate of AML transformation. Rates of hematological improvement with the use of azacitidine were lower than expected. Lenalidomide is an effective treatment option and should be considered when treating patients with RARS-T. [Display omitted] DisclosuresSallman:Celgene: Research Funding, Speakers Bureau. List:Celgene: Research Funding. Komrokji:Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau.

Impact of Red Cell Distribution Width on Dysplasia and Clinical Outcome in 66 Patients with Myelodysplastic Syndrome without Increased Blasts

Both clonal hematopoiesis as defined by genomic alteration and morphological cell dysplasia is a hallmark of pathology of myelodysplastic syndrome (MDS). Studies showed that, red cell distribution width (RDW), a quantitative non-subjective index of red cell anisocytosis, has diagnostic significance in anemia associated with MDS.Our recent study showed that increase of RDW has close association with dyserythropoiesis and also shorter overall survival (OS) in refractory anemia (Leuk Res 2018;67:56-59). To explore the clinical significance of RDW in MDS without increased blasts, we examined the details of correlation between RDW and morphological dysplasia which characterize MDS.We retrospectively analyzed 101 MDS patients, including 66 patients without increased blasts (blasts in the bone marrow of less than 5% and the peripheral blood of 1% or less). RDW was calculated using automated blood cell analyzer. Dysplasia was assessed according to the WHO 2008 classification by different two investigators. 100 cells (at least 25 cells) of myeloid and erythroid series were counted. At least 25 cells of megakaryocyte were also counted. Karyotype of bone marrow cells was analyzed by using routine G-band technique and categorized according to MDS cytogenetic scoring system. Correlations of RDW and clinical variables were analyzed. Based on previous study, MDS patients was divided into two groups, RDW-H (RDW was 15% or more, 48 cases) and RDW-L (RDW was lower than 15%, 18 cases). Comparisons of dysplasia or OS between these two groups were analyzed.In MDS without increase of blasts, weak inverse correlation was found between RDW and hemoglobin (rs = −0.31, P = 0.01). RDW was correlated with the presence of ring sideroblasts (RS) in the bone marrow (rs = 0.40, P = 0.001), and also weakly correlated with budding of nucleus of erythroblasts (rs = 0.33, P = 0.007). Dysgranulopoiesis was less observed than either dyserythropoiesis or dysmegakaryopoiesis (P <0.001, P <0.001). RDW was not significantly different between groups with karyotype risk category (P = 0.08).Table showed the subtype of MDS, details of morphological dysplasia. Between two groups, RDW-H showed significantly higher percentages of dyserythropoiesis, dysgranulopoiesis, and dysmegakaryopoiesis.The increased RDW (≥15.0%) was associated with shorter OS (P = 0.02) (hazard ratio, 1.15e + 9 (95% confidence interval = 2.49-2.49)) (Figure). In contrast, there was no association between RDW and OS in MDS patients with increased blasts.Earlier studies showed increase of RDW was evident in refractory anemia with ring sideroblasts (RARS) which showed erythrocyte anisocytosis called dimorphism. Revised WHO classification (2017) defined MDS-RS with single lineage dysplasia (MDS-RS-SLD) which is characterized with specific mutation of splicing factors, such as SF3B1 and with favorable prognosis. This classification also defined MDS-RS with multi lineage dysplasia (MDS-RS-MLD) which has additional dysplasia in myeloid and/or megakaryocyte lineage in addition to RS. Pathology of MDS-RS-MLD is still to be remained.Our findings partly agree the earlier observations, showing close association between red cell anisocytosis evaluated with increase of RDW and the presence of RS. Furthermore, RDW-H group contained MDS with dysplasia of myeloid and/or megakaryocyte lineage not limited to the erythroid dysplasia(s) with RS. RDW could discriminate MDS-MLD with poor prognosis from MDS-SLD with favorable prognosis.To explore the significance of RDW in MDS, it is necessary to study large number of patient combined with genetic analysis. Nevertheless, RDW is potentially simple and useful laboratory parameters in clinical practice in MDS, representing hematopoietic defect (ineffective hematopoiesis), associated with iron metabolism and hemoglobin synthesis in MDS. DisclosuresNo relevant conflicts of interest to declare.

Percentage of Erythroid Cells in Bone Marrow at Diagnosis As a New Prognostic Factor in Patients with Myelodysplastic Syndrome

IntroductionRisk assessment is essential for guiding therapy in patients with myelodysplastic syndromes (MDS). Currently, the most widely used prognostic scoring models are the International Prognostic Scoring System (IPSS) and the revised IPSS (IPSS-R). The prognostic relevance of the percentage of erythroid precursors (EP) in bone marrow (BM) and its relationship with other biological characteristics has been poorly studied, although it has been proposed that a very low percentage of EP in BM may represent a cohort of patients with potentially adverse outcome. Our main aim was to analyze the biological and clinical features of MDS patients according to the percentage of EP in BM at diagnosis and evaluate its prognostic value on survival.Patients and MethodsData from 4,791 de novo MDS patients from the MDS Spanish Registry with available cytogenetics were collected. All patients included were diagnosed based on the 2008 WHO criteria and risk stratification was performed following the IPSS-R. Patients were distributed, according to the percentage of EP in BM, into three groups: less than 15% (EP<15%), between 15% and 49% (EP 15-49%) and equal or more than 50% (EP>49%).Proportions were compared by the Chi-square test. Survival curves were constructed by Kaplan-Meier method and differences between curves were evaluated by log rank tests. Multivariable analysis of survival was performed using Cox's proportional hazards regression model. P-values <0.05 were considered as statistically significant. All statistical analyses were performed by the R software.ResultsThe 4,791 cases were grouped according to the percentage of erythroid cells in BM at diagnosis. In Table 1 are displayed the clinical and biological characteristics of the patients in the whole series and in the three groups based on the EP percentage in BM. The group with EP<15% in BM showed a significantly greater number of cytopenias at diagnosis, a higher number of peripheral blood (PB) and BM blasts, and a higher percentage of patients classified in MDS subtypes with excess blasts. The EP>49% group presented higher rates of patients with refractory anemia with ring sideroblasts (RARS), lower number of PB and BM blasts, and more frequent high-risk cytogenetics. Conversely, the intermediate EP group with EP 15-49% showed less number of cytopenias, higher frequency of good-prognosis cytogenetics, and lower percentage of patients in higher-risk IPSS-R categories.Survival analyses confirmed that age, blast percentage in PB and BM, and IPSS-R cytogenetics are significantly associated with worse survival. A striking difference in survival within groups according to percentage of EP was also found, recognizing EP percentage as an independent risk factor for survival of MDS patients in multivariate analysis. Patients with EP<15% in BM experienced a significantly shorter OS (median, 28 months) in both univariable (P<0.0001) and multivariable analysis (P<0.0001) (Table 2 and Figure 1). The best survival was found for patients with EP 15-49% with a median survival of 58 months. The group with EP>50% in BM had also worse survival than the intermediate group, showing a median survival of 47 months (hazard ratio, 0.79; 95% CI, 0.67 to 0.93; P < 0.005).ConclusionsOur study proves the prognostic impact of the percentage of erythroid precursors in bone marrow in MDS patients, providing evidence that the presence of EP <15% in BM is an independent risk factor for survival. [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 Novel Metabolic Transcriptome Identified in Myelodysplastic Syndromes (MDS) Correlates with OMS Classification and Poor Prognosis

Myelodysplastic syndromes (MDS) are clonal malignancies of the hematopoietic stem cell leading to an ineffective hematopoiesis with a complex and poorly understood pathophysiology combining increased apoptosis and propensity to transformation associated with immune dysregulation. Despite a major improvement of the classification of MDS in terms of diagnosis and prognosis according to OMS recommendations, a significant fraction of MDS remains unclassified. In this study, we wished to determine if the integrative analysis of global MDS transcriptome associated with single cell experiments performed on CD34+ hematopoietic progenitors using novel bioinformatics tools could identify novel signaling pathways associated with different subtypes of MDS. This transcriptomic analysis included a large cohort of of MDS patients (n=323) as compared to healthy controls (n=86). We concentrated our analysis in the pathways involved in metabolomics as there was an increase of expression of genes involved in metabolic pathways in MDS transcriptome as compared to hematopoietic progenitors from healthy donors. Metabolic meta-analysis on HP transcriptome (datasets GSE15061 and GSE58831) were subsequently analyzed by creating a software “gene2bcp.sh” (https://github.com/cdesterke/gene2bcp) based on metabolic database “BioCycPathway”. Forty two genes involved in metabolism were found to be upregulated in MDS samples (LIMMA algorithm, FDR adjust p-value < 0.05 and fold change > 2 in both dataset). A multivariate model was built by selecting the best 17 genes and in order to validate these findings an independent cohort including 11 healthy donors and 55 MDS patients (pts) were studied. This latter group included 18 patients with refractory anemia (RA), 19 pts with RA with ring sideroblasts (RARS) and 18 pts with RA with excess of blasts (RAEB) (GSE4619). This analysis (fig1A) allowed to predict MDS diagnosis as compared to healthy donors in a robust way (p=0.0013). Similarly with this unique metabolic transcriptome, we could stratified MDS patients correctly according to their OMS classification (p=0.00065). Metabolic transcriptome also clearly identified patients with MDS with cytogenetic abnormalities, in particular MDS with 5q- syndromes as compared to MDS without cytogenetic abnormalities (p=0.0023). We then applied these results to the RNAseq single-cell sequencing with cytometric analysis of a MDS patient with RAEB2 and monosomy 7 (GSE99095). Single cell trajectories were built based on 39 metabolic genes identified from previous meta-analysis. Monocle2 algorithm with metabolic gene profile allowed to discriminate CD34+CD38- from CD34+CD38+ cells after “tSNE” clustering (fig1B). These results were submitted to a pseudotime analysis which allows the analysis of transitional states in cell signaling pathways. Nine transition stages were found with stage 5 enriched in CD34+CD38- and stages 1 and 7 were found enriched in CD34+CD38+ (fig1C), differential test on this metabolic trajectory stratified genes in 6 clusters (gig 1D), with an enriched high expression of ACOT4 (Acyl-CoA Thioesterase 4), EARS2 (Glutamyl-TRNA Synthetase 2), PLD6 (Phospholipase D Family Member 6), PSAT1 (Phosphoserine Aminotransferase 1) in sub-compartment CD34+CD38- of this RAEB2 MDS patient. Overall, we show here for the first time a novel metabolic transcriptome correlating with classical MDS subtypes and prognosis. We describe a primitive metabolic trajectory in single bone marrow cells able to reconstitute the clonal architecture. The genes that we identified could be of use as additional markers of diagnosis and prognosis in future studies. DisclosuresNo relevant conflicts of interest to declare.

SF3B1 deficiency impairs human erythropoiesis via activation of p53 pathway: implications for understanding of ineffective erythropoiesis in MDS

BackgroundSF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown.MethodsshRNA-mediated approach was used to knockdown SF3B1 in human CD34+ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1.ResultsWe document that SF3B1 knockdown in human CD34+ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts.ConclusionsThese findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

Efficacy of Lenalidomide in Myelodysplastic/Myeloproliferative Neoplasms with Ring Sideroblasts

Background: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ring sideroblasts and thrombosis (MDS/MPN-RS-T), include the dysplastic features of Refractory Anemia with Ring Sideroblasts (RARS) and the myeloproliferative features of essential thrombocythemia (ET) and are characterized by a high rate (50%) of JAK2 V617F mutations or rarely by the presence of mutations in exon 10 of the MPL (myeloproliferative leukemia) gene or in exon 9 of the calreticulin gene. We previously demonstrated that RARS-T differed from RARS and ET from a clinical, biological and prognostic point of view and that the presence of high rates of splicing factor 3B subunit 1 (SF3B1) mutations in RARS-T and the absence of these mutations in ET strengthened the hypothesis that RARS-T was a distinct entity. We also reported in a large series of patients (n=200) that the risk of thrombosis was higher in RARS-T than in RARS patients without a high platelet count, leading clinicians to reduce the platelet count in almost 33% of RARS-T patients. However, due to the presence of anemia, the use of the cytoreductive agents usually used in ET is limited since they worsen the cytopenia. This explains why it is difficult to treat patients who harbor both anemia and thrombocytosis. Lenalidomide, an immunomodulatory agent frequently used in low-risk MDS, has very rarely been tested in MDS/MPN-RS-T patients, with conflicting results. Objective : To test the effect of Lenalidomide on MDS/MPN-RS-T Patients: Here, we report our experience using lenalidomide in two women who presented with JAK2 V617F negative MDS/MPN-RS-T. Results: The first patient was a 78 year-old woman, with a complete blood count (CBC) showing hemoglobin 80 g/L, platelet count 686 G/L and leukocytes 6 G/L. The second was a 58 year-old woman whom initial CBC showed hemoglobin 113 g/L, platelet count 1,600 G/L, leukocytes 8.2 G/L. In both cases the bone marrow aspirates showed erythroid hyperplasia with myelodysplasic features and 64% and 24% of ring sideroblasts respectively, associated with marked dysmegakaryopoiesis, leading to the diagnosis of MDS/MPN-RS-T. No excess of blast cells was noted. Bone marrow cytogenetics showed in both cases a normal 46XX karyotype. A Lys700Glu SF3B1 mutation was noted for one patient (not tested for the second), without the JAK2 V617F or MPL exon10 or CALR mutations. In the first patient, blood transfusions were initiated for one year, however, due to an increase in dependence on red blood cell transfusions, treatment with lenalidomide (5 mg daily 21/28days) was started and led to a decrease in the platelet count from 686 to 220 G/L associated with an regular improvement in hemoglobin levels from 80 to 100 g/L within the first 28 weeks of lenalidomide (only 6 units of red blood cells were transfused along this period). Finally, no transfusion was required in the subsequent 47 weeks since the hemoglobin level was above 90 g/L. However, due to a low hemoglobin level, red blood cell transfusions were reinitiated, but with a lower frequency (2 units of red blood cells every 2 months), in association with lenalidomide. Altogether, over the 20 months of treatment with lenalidomide, the red blood cell requirement was drastically reduced. However, grade IV neutropenia was observed (granulocytes 0.5 G/L) but without any infectious disease, leading to stop the treatment. In the second patient, a watch-and-wait strategy was initiated, but due to an increase in the platelet count > 2,000 G/L, hydroxyurea (500 mg/day) was started, worsening the anemia to 108 g/l then 84 g/l leading to the use of erythropoietin once/week. Hydroxyurea was then stopped due to its adverse effects on hemoglobin, leading to the increase in the platelet count to 3,622 G/L. A treatment with lenalidomide (5 mg daily 21/28days) was then started and secondarily improved to 10 mg daily 21/28days since very well tolerated. A marked decrease in the platelet count was noted, from 3,622 to 985 G/L whereas hemoglobin level rised up from 92 to 115 g/L. Off note, for this patient erythropoietin treatment was maintained every 2 weeks in combination with lenalidomide. Conclusion: This two-cases experience confirms the partial efficacy of lenalidomide in MDS/MPN-RS-T in normalizing (case 1) or markedly reducing (case 2) the platelet count and allowing of independence from blood transfusion (at least for 10 months in case 1). Disclosures No relevant conflicts of interest to declare.

Gene mutations from 511 myelodysplastic syndromes patients performed by targeted gene sequencing

目的探索中国骨髓增生异常综合征（MDS）患者基因突变特征。方法采用包含112个血液肿瘤相关基因的靶向测序技术对511例MDS患者进行基因突变检测，并回顾性分析其临床资料。结果全部511例MDS患者中，共检测到83个突变基因，439例（86%）患者伴有至少1个基因突变。对涉及的83个基因依据其功能进行分类，突变频率依次为表观遗传相关基因（50%）、剪接子相关基因（37%）、信号传导相关基因（34%）、转录因子（24%）和细胞周期与凋亡相关基因（17%）。不同亚型患者的平均基因突变数目分别为：难治性血细胞减少伴单系发育异常（RCUD）1.25个，难治性血细胞减少伴多系发育异常（RCMD）1.73个，难治性贫血伴环状铁粒幼红细胞（RARS）2.79个，难治性贫血伴原始细胞增多-1（RAEB-1）2.22个，RAEB-2 2.34个，MDS伴单纯5q− 2.67个，MDS未分类（MDS-U）2.00个。U2AF1突变患者单纯+8核型异常比例显著增高[Q<0.001，OR=4.42（95% CI 2.23~8.68）]，复杂核型比例显著减低[Q=0.005，OR=0.22（95% CI 0.04~0.72）]。依据基因突变数目，将患者分为伴有0~1个基因突变组，伴有2个基因突变组和伴有3个及以上基因突变组，三组总生存（OS）差异具有统计学意义（P=0.041）。结论近九成的MDS患者伴有至少1个基因突变，表观遗传相关基因和剪接子相关基因是MDS最常受累基因。随着疾病的演进基因突变数增多，基因突变数目多的患者OS较差。

Transcriptome analysis of CD34+ cells from myelodysplastic syndrome patients

The myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal hematologic stem cell disorders with the characteristic of ineffective hematopoiesis leading to low blood counts, and a risk of progression to acute myeloid leukemia (AML). To understand specific molecular characteristics of different MDS subtypes with del(5q), we analyzed the gene expression profiles of CD34+ cells from MDS patients of different databases and its enriched pathways. 44 genes, such as MME and RAG1, and eight related pathways were identified to be commonly changed, indicating their conserved roles in MDS development. Additionally, U43604 was identified to be specifically changed in three subtypes with del(5q), including refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS) and refractory anemia with excess blasts (RAEB). C10orf10 and CD79B were specifically changed in RA patients with del(5q), while POU2AF1 were in RARS patients with del(5q). We also analyzed specific pathways of MDS subtypes, such as “Glycosaminoglycan biosynthesis-chondroitin sulfate” which was specific identified in RARS patients. Importantly, those findings can be validated well using another MDS database. Taken together, our analysis identified specific genes and pathways associated with different MDS subtypes with del(5q).

DNA repair gene expressions are related to bone marrow cellularity in myelodysplastic syndrome

ObjectiveTo evaluate the expression of genes related to nuclear excision (ERCC8, XPA and XPC), homologous recombination and non-homologous end-joining (ATM, BRCA1, BRCA2 and LIG4) repair mechanisms, using quantitative PCR methodologies,...

Interaction between ring sideroblasts and treatment with hypomethylating agents (HMA) in patients with refractory anemia with ring sideroblasts.

e18559 Background: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms defined by ineffective hematopoiesis, dysplastic morphologic features, and variable risks of progression to acute myeloid leukemia (AML). Refractory anemia with ring sideroblasts (RARS) is a subtype of MDS defined by &lt; 5% basts and &gt; 15% ring sideroblasts (per 2008 WHO). Because RARS is a lower risk disease which does not frequently require HMA therapy, very little is known about the effects of HMA on RARS. Methods: A total of 1325 MDS patients’ data from 1993-2016 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our center. Patients were considered for our study if they received HMA for their RARS. Response was identified based on MDS IWG 2006 criteria. Prognostic factors were analyzed by univariate and multivariate analyses. Survival estimates were calculated using Kaplan-Meier curves. Results: 168 of the initial 1325 patient were RARS (14%); only 14 (8%) were treated with HMA. Median age was 72 years (range, 51-85); half were males, with median overall survival of 91.3 months. The median number of ring sideroblasts (RS) at diagnosis was 40% (range 10-85%). Of the 14 patients, 11 (79%) received azacitidine (AZA), 2 (14%) decitabine (DAC) and one received both (7%). The median number of cycles for AZA was 6.5 (range, 2-28) vs 6 cycles for DAC (range, 1-24). Of the 14 patients receiving HMA, only 3 responded (21%) achieving hematologic improvement. All 3 responders received AZA. 6 of the 14 RARS patients (43%) had their bone marrow biopsy repeated after HMA therapy, 3 of whom were among the responders to AZA therapy and 3 were non-responders. In 5 patients, ring sideroblasts decreased after HMA therapy by a median of 15% (range 0-80); they remained the same in 1 patient; one patient who had developed AML had a decrease in RS from 85% to 5%. Conclusions: RARS is a low-risk subtype of MDS that is infrequently treated with HMA (8% in our study). Response rate to HMA (21%) was inferior to known rates in MDS. HMA therapy seems to decrease the number of ring sideroblasts irrespective of response. RARS patients should be treated on clinical trials if available.

Clinical significance of HFE gene mutations in patients with refractory anemia with ring sideroblasts (RARS).

e18556 Background: RARS is a subtype of myelodysplastic syndromes (MDS) defined by &lt; 5% blasts and ≥15% ring sideroblasts (WHO 2008). Hereditary hemochromatosis is a disorder characterized by dysregulations in iron absorption, largely associated with C282Y and H63D mutations of the HFE gene. Iron levels are elevated in both disorders and pathophysiologic correlations were suggested. HFE gene mutations were previously found higher in MDS compared to controls (50% vs 36%) ( Nearman et al, Am J Hematol 2007). Methods: A total of 168 RARS patients’ data from 1994 to 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our center. We searched patients’ records retrospectively to Identify those tested for HFE gene (C282Y, H62D, S65C) mutations, done inside or outside our institution. Survival estimates were calculated using Kaplan-Meier curves. Results: Out of the 168 RARS patients, only 17 (10%) were tested for HFE gene mutations. Out of the 17 tested, 11 (65%) were found to have mutations; 2 of which (18%) had homozygous H63D mutation, 1 patient (9%) had double heterozygous H63D and C282Y mutations, 5 (45%) had only one H36D heterozygous mutation vs 3 patients (27%) with only one C282Y heterozygous mutation. Only one patient was tested for the additional S65C mutation and it was not detected. H63D mutation was present in a total of 8 patients (73%) vs C282Y mutation which was present in 4 patients (36%). Bone marrow iron stores were increased in all 17 tested patients, except one who had decreased stores, this patient had one heterozygous C282Y mutation. Median overall survival (mOS) was 117 months in the HFE mutated patients vs 75 months in the non-mutated (p = 0.6). Conclusions: Our study found the HFE gene, when tested, to be mutated in higher frequencies among patients with RARS compared to that reported in the general population (65% vs 36%), with H63D mutation in almost three quarters of all mutated patients. Although it did not reach statistical significance, the longer survival observed among HFE mutated patients compared to the wild-type raises the question whether testing for HFE gene mutations among patients with MDS-RARS should be further explored.