Prognostic interaction between bone marrow morphology and SF3B1 and ASXL1 mutations in myelodysplastic syndromes with ring sideroblasts

Abstract Introduction Splicing factor (SF) mutations represent a novel class of driver mutations in myelodysplastic syndromes (MDS), where SF3B1 and SRSF2 are most frequently affected. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets whose abnormal splicing is responsible for the pathogenesis of MDS. Methods We enrolled a total of 480 patients with MDS, for whom complete clinical and pathological data were available. RNA sequencing was performed for bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells from 215 MDS patients. Observed splicing junctions were compared between samples with and without each SF mutation. In SF-mutated cases, NMD could cause severe degradation of abnormal transcripts and obscure the effect of SF-mutants. To sensitively detect abnormal transcripts otherwise degraded by nonsense-mediated RNA decay (NMD), analysis was also performed on BM/MNCs from 7 patients and CD34+ bone marrow cells from 3 patients with or without inhibition of NMD by cycloheximide (CHX). Common mutations and copy number variations were also investigated using targeted sequencing. Results SF3B1 and SRSF2 mutations were associated with distinct clinical phenotypes and outcomes. SF3B1-mutated cases typically showed isolated erythroid dysplasia and high proportion of ring sideroblasts, whereas SRSF2 mutations correlated with a significantly higher incidence of myeloid and megakaryocyte dysplasia (P<.001), higher proportion of bone marrow blasts (P=.02) and lower degree of erythroid dysplasia and proportion of ring sideroblasts (P<.001). SF3B1- and SRSF2-mutated myeloid neoplasms were also associated with a significantly different outcome, SRSF2-mutated neoplasms having a significantly shorter survival (HR=5.35, P<.001). To explore the molecular basis of these distinct features in terms of splicing defects, RNA sequencing data from SF3B1-mutated (n = 68) and SRSF2-mutated (n = 39) BM/MNCs and CD34+ cells were compared with those without known SF mutations (n = 91) to detect splicing events significantly enriched in SF-mutated cells. In total 748 and 589 splicing events were enriched in SF3B1- and SRSF2-mutated samples. Among these, 203 (27%) of SF3B1-specific events were observed almost exclusively in SF3B1-mutated samples;193 (95%) were caused by misrecognition of 3' splice sites of which ~50% resulted in frameshift. In contrast, in SRSF2-mutated cases, the predominant defects were alternative exon usage, which accounted for for 80% of the abnormalities. However, the effect of mutant-SRSF2 on abnormal splicing was generally small, with 89% showing only <3× more abnormal transcripts in SRSF2-mutated. Similar results were obtained for BM/MNCs for both mutations. Splicing defects of both SF-mutations involved substantially different set of genes. Aberrant splicing enriched in SF3B1- and SRSF2-mutated samples involved 12 and 7 cancer-related genes defined by the Cancer Gene Census with no genes overlapped in between. Of special interest among these was EZH2, which showed 2 SRSF2-associated alternative exons with a premature termination codon. EZH2 transcripts having these exons are expected to be susceptible to NMD-mediated degradation. A similar defect was also detected in another component of the polycomb repressive complex 2 (PRC2), implicated in compromised function of PRC2 in SRSF2-mutated cases. On the other hand, 2 genes involved in mitochondrial heme synthesis were significantly affected in SF3B1-mutated samples. In addition, an additional gene engaged in heme synthesis, ABCB7, was identified from experiments using NMD inhibition to detect 'masked splicing'. ABCB7 is one of the causative genes for congenital sideroblastic anemia and uniformly showed reduced expression in SF3B1-mutated samples, most likely due to abnormal splicing. Conclusion SF3B1 and SRSF2 mutations have distinct impacts on clinical phenotypes and outcomes together with RNA splicing. SF3B1 mutations caused misrecognition of 3' splice sites, frequently resulting in truncated gene products and/or decreased expression due to NMD. SRSF2 mutations were characterized by modest but more widespread alterations in exon usage of genes including multiple components of PRC2. Our results provide insights into the pathogenesis of SF-mutated MDS. Disclosures No relevant conflicts of interest to declare.

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Spontaneous and cytokine-induced thrombocytopenia in myelodysplastic syndromes: serum thrombopoietin levels and bone marrow morphology

British Journal of Haematology ◽

10.1046/j.1365-2141.1999.01442.x ◽

1999 ◽

Vol 105 (4) ◽

pp. 966-973 ◽

Cited By ~ 11

Author(s):

Eva Hellström-Lindberg ◽

Lena Kanter-Lewensohn ◽

Janet Nichol ◽

Åke Öst ◽

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndromes ◽

Bone Marrow Morphology

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SF3B1 Mutations Are Associated with Favorable Cytogenetics in Patients with Myelodysplastic Syndromes with Ring Sideroblasts

Blood ◽

10.1182/blood.v120.21.3823.3823 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3823-3823 ◽

Cited By ~ 1

Author(s):

Vincent-Philippe Lavallée ◽

David Faucher ◽

Bruno Lamontagne ◽

Josée Hébert ◽

Lambert Busque

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndromes ◽

World Health ◽

Refractory Anemia ◽

Wild Type ◽

Event Free Survival ◽

Free Survival ◽

Mutational Hotspots ◽

Ring Sideroblasts ◽

Bone Marrow Blasts

Abstract Abstract 3823 Background: Somatic mutations of splicing factor 3B, subunit 1 (SF3B1) are prevalent in myelodysplastic syndromes (MDS) with ring sideroblasts (RS). Little is known about the clinical phenotype associated with these mutations. Initial studies have reported an excellent event-free survival (EFS) in patients with SF3B1 mutations but subsequent reports did not confirm that observation. Therefore, its clinical significance remains uncertain. Our objective was to determine if the SF3B1 mutational status predicts distinctive morphological, cytogenetic or clinical characteristics in patients with MDS with RS. Methods and patients: Twenty-seven patients diagnosed with MDS and ≥ 15% RS in bone marrow aspiration between 2000 and 2011 at our institution and other hospitals affiliated to the Quebec Leukemia Cell Bank (BCLQ) and with available material for genetic analyses were included in this study. Morphology of samples was revised. After DNA extraction, polymerase chain reaction was performed to amplify SF3B1 mutational hotspots in exons 13 to 15. Amplicons were sequenced by the Sanger method. Clinical and laboratory data were collected retrospectively from hospital medical records and BCLQ database. Results: Point mutations of SF3B1 were found in 48% (13/27) with the following distribution: refractory anemia with RS (RARS): 7/10, refractory cytopenia with multilineage dysplasia (RCMD): 5/10 and refractory anemia with excess blasts (RAEB): 1/7. All mutations were heterozygous nonsense mutations and K700E exon 15 mutations accounted for 85% (11/13) of mutations. The remaining mutations were located in exon 14 at position 666. In the entire cohort, SF3B1 mutations were associated with an older median age at diagnosis (69 vs 60 years, p= 0.007). They were associated with favorable cytogenetics whereas unmutated SF3B1 was associated with unfavorable cytogenetics (8 cases vs 0 with favorable cytogenetics and 0 cases vs 11 with unfavorable cytogenetics for mutated and wild type SF3B1 respectively, p < 0.0001). Median overall survival (OS) was similar in the 2 groups (not reached vs 27 months, p=0.7) but median EFS was higher in patients with mutated SF3B1 (not reached vs 5 months, p=0.02). To evaluate the influence of higher bone marrow blasts on outcomes, analyses were restricted to the RARS and RCMD-RS categories. In that subgroup, SF3B1 mutations were correlated with higher median platelet counts at diagnosis (201 vs 70 × 109/L, p=0,04). In addition, SF3B1 mutations remained associated with favorable cytogenetics, and unmutated SF3B1 with unfavorable cytogenetics (p=0.004). Moreover, SF3B1 mutations were more frequently detected within favorable or intermediate-1 categories of the International Prognostic Scoring System (IPSS) compared to wild type SF3B1 (p=0.04). However, in patients with < 5% bone marrow blasts, EFS was similar between mutated and unmutated SF3B1 (not reached vs 46 months, p =0.4). Conclusion: In this series, SF3B1 mutations are frequent in MDS with RS and they are strongly associated with favorable cytogenetics. They are mostly detected among low-risk groups of the World Health Organisation classification (RARS and RCMD-RS), but they are not independently associated with an increased overall or event-free survival. Disclosures: No relevant conflicts of interest to declare.

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Molecular Genetic Analysis of Myelodysplastic Syndromes (MDS) Patients with Ring Sideroblasts (RS); Independent Confirmation of Association of SF3B1 Mutations with Better Prognosis

Blood ◽

10.1182/blood.v124.21.3237.3237 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3237-3237

Author(s):

Abdullah Mahmood Ali ◽

Nicholas Iverson ◽

Alexander Penson ◽

Allison Scott ◽

Tabish Riaz ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Genetic Analysis ◽

Myelodysplastic Syndromes ◽

Molecular Genetic ◽

Jak2 V617f ◽

Molecular Genetic Analysis ◽

Kaplan Meier ◽

Ring Sideroblasts ◽

Calr Mutations

Abstract Background: Ring sideroblasts (RS) are erythroblasts with iron-loaded mitochondria, appears as blue granules when stained with Prussian blue, and are characteristic of myelodysplastic syndromes (MDS) subgroups refractory anemia with ring sideroblasts (RARS), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), and RARS with thrombocytosis (RARS-T) but are also infrequently found in other MDS groups. The etiology of RS remains unclear. Molecular genetic analysis of MDS patients with RS (MDS-RS) using exome and targeted sequencing identified mutations in several genes including SF3B1. While the RS phenotype was shown to be strongly associated with SF3B1 mutations, the association of SF3B1 mutations with better prognosis remained controversial. Also, there remained several patients who do not show any mutations in SF3B1 suggesting there are additional genes associated with RS phenotype. In addition to SF3B1 mutations, RARS-T patients show mutations in JAK2 (V617F and Exon 12), MPL and CALR genes, frequently mutated in Essential Thrombocytopenia (ET). But there remained a high number of patients who do not show any mutation in JAK2, MPL and CALR genes suggesting additional genes are associated with thrombocytosis. In order to gain further insight into the molecular genetics of MDS-RS patients and to elucidate their clinical significance we screened a large cohort of patients with RS (RARS/RCMD-RS) and a subgroup with thrombocytosis (RARS-T). Methods: This study is approved by the Institutional Review Board of Columbia University and informed consent was obtained from all the individuals participated in the study. Bone marrow mono nuclear cells were isolated from bone marrow aspirate and DNA was extracted using DNeasy Blood and Tissue kit. To screen for most frequent mutations in SF3B1 (exon 14 and 15), MPL (exon 10), JAK2 (exon 12), and CALR (exon 9) genes, primers were designed to amplify the exons and the exon-intron junctions using PCR and the amplified and purified PCR products were sequenced using Sanger sequencing on both strands. To screen for mutations V617F in JAK2, primers were designed to carry out allele specific PCR. Demographic and clinical data is collected from patient’s charts/reports. Statistical analysis including survival curve (Kaplan–Meier method) was performed using GraphPad Prism Software. Results: A total of 209 patients with RS phenotype were screened for SF3B1 mutations. Mutations in SF3B1 gene were detected in 62% (130/209) of RS patients, including 85% (23/27) of RARS-T and 59% (107/182) of RARS/RCMD-RS patients. Among all the SF3B1 mutations, K700E was the most frequent mutation (60%) followed by mutations at H662. Clinical significance of SF3B1 mutations on overall survival, using Kaplan-Meier method, showed SF3B1 mutations were associated with better prognosis (Fig 1). The median survival of patients with SF3B1 mutation is 110 months compared to those without mutations, 70 months (p value < 0.034). Studies to screen SF3B1 negative patients for additional mutations are being carried out using exome sequencing to identify genes associated with RS phenotype. In addition to SF3B1, RARS-T patients were also screened for JAK2 V617F and exon 12 mutations. JAK2 V617F mutations were detected in 11% of RARS-T patients and no mutations were found in JAK2 exon 12. Patients negative for JAK2 were screened for mutations in MPL and CALR genes. No mutations were found in MPL but 8% (2/23) of those negative for JAK2 were found to have CALR mutations. Both CALR mutations were frameshift mutations that alter the C-terminus thus abolishing the KDEL sequence required for CALR function. There are still 80% RARS-T patients in our cohort who do not show any mutations in JAK2, MPL, CALR genes. Conclusions: The association of SF3B1 mutation with prognosis is controversial with some studies suggesting a better prognosis while other reports no effect. Our study, using a large cohort of well-characterized RS patients provides an Independent verification of the observation that SF3B1 mutations are associated with better overall survival. There remains a large group of patients (40% in RARS/RCMD-RS groups) without SF3B1 mutation but with a RS phenotype suggesting yet other gene(s) is associated with RS phenotype. Similarly, there remain patients who do not show any mutations in JAK2, MPL and CALR suggesting other genes are involved in the etiology of thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Acquired Elliptocytosis as a Manifestation of Myelodysplastic Syndrome with Ring Sideroblasts and Multilineage Dysplasia

Case Reports in Hematology ◽

10.1155/2017/3625946 ◽

2017 ◽

Vol 2017 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Jacob D. Kjelland ◽

Denis M. Dwyre ◽

Brian A. Jonas

Keyword(s):

Bone Marrow ◽

Chest Pain ◽

Myelodysplastic Syndrome ◽

Bone Marrow Biopsy ◽

Myelodysplastic Syndromes ◽

Rare Case ◽

Elderly Male ◽

Multilineage Dysplasia ◽

Ring Sideroblasts

Acquired elliptocytosis is a known but rarely described abnormality in the myelodysplastic syndromes (MDS). Here we report the case of an elderly male who was admitted to the hospital with chest pain, dyspnea, and fatigue and was found to be anemic with an elliptocytosis that had only recently been noted on peripheral smears of his blood. After bone marrow biopsy he was diagnosed with MDS with ring sideroblasts and multilineage dysplasia and acquired elliptocytosis. Here we report a rare case of acquired elliptocytosis cooccurring with MDS with ring sideroblasts and multilineage dysplasia.

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The Prognostic Evaluation of Myelodysplastic Syndromes with Ring Sideroblasts can be Improved by Considering Bone Marrow Blasts from Nonerythroid Cells

Leukemia Research ◽

10.1016/s0145-2126(17)30348-x ◽

2017 ◽

Vol 55 ◽

pp. S135-S136

Author(s):

L. Arenillas ◽

X. Calvo ◽

E. Luño ◽

L. Senent ◽

M. Arnan ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndromes ◽

Prognostic Evaluation ◽

Ring Sideroblasts ◽

Bone Marrow Blasts

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SF3B1 Mutations Are Prevalent in Myelodysplastic Syndromes with Ring Sideroblasts but Do Not Hold Independent Prognostic Value

Blood ◽

10.1182/blood.v118.21.460.460 ◽

2011 ◽

Vol 118 (21) ◽

pp. 460-460 ◽

Cited By ~ 2

Author(s):

Mrinal M. Patnaik ◽

Terra L Lasho ◽

Janice M Hodnefield ◽

Ryan A Knudson ◽

Rhett P Ketterling ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndromes ◽

Prognostic Value ◽

Univariate Analysis ◽

Multivariable Analysis ◽

World Health ◽

Refractory Anemia ◽

Ring Sideroblasts ◽

Risk Categories ◽

Good Risk

Abstract Abstract 460 Background: SF3B1 mutations were recently reported to occur in myelodysplastic syndromes (MDS), especially in the presence of ring sideroblasts (RS) (Papaemmanuil et al. Leukemia Research 2011, Volume 35, Supplement 1, page S18). We sought to accurately define the interaction between SF3B1 mutations, bone marrow morphology, karyotype and prognosis in MDS with ≥15% RS (MDS-RS). Methods: Mayo Clinic databases and cell banks were queried to identify patients with MDS and ≥15% RS (MDS-RS). All study patients were required to have undergone bone marrow examination and cytogenetic evaluation at diagnosis. Pathology slides, including iron stains, were centrally re-reviewed to accurately quantify bone marrow RS percentage and confirm World Health Organization (WHO) morphologic categories. Detailed analysis of clinical and cytogenetic parameters was performed to risk stratify patients according to the revised International Prognostic Scoring System (IPSS-R) (Greenberg et al. Leukemia Research 2011, Volume 35, Supplement 1, page S6). All patients were annotated for their mutational status involving JAK2, MPL and IDH. PCR sequencing was used to identify SF3B1 mutations. Results I: Baseline patient information: The study is currently ongoing; to date, results are availab1e in 88 patients (median age 72 years, 69% males) including 36 (41%) patients with refractory anemia with RS (RARS), 36 (41%) with refractory cytopenia with multilineage dysplasia and ≥15% RS (RCMD-RS), 11 (12%) with refractory anemia with excess blasts (RAEB)-1 and ≥15% RS (RAEB1-RS) and 5 (6%) with RAEB2-RS. Median (range) values were 9.1 g/dL (5.8–15.7) for hemoglobin, 4.1 × 109/L (1.2–35.2) for leukocytes and 164 × 109/L (6–585) for platelets. IPSS-R risk categories were 20% very good, 49% good, 11% intermediate, 3% poor and 16% very poor. Karyotype was normal in 60%, other very good/good risk 16%, intermediate-risk 2%, and poor/very poor in 22%; 14% had monosomal karyotype (MK). Six (7%), four (5%) and zero (0%) patients displayed IDH, JAK2 or MPL mutations. Red cell transfusion need was documented in 27 (31%) patients. Results II: Prevalence and distribution of SF3B1 mutations and clinical correlates: SF3B1 mutations (all heterozygous) were detected in 40 (∼46%) of all 88 study patients: 25 (63%) K700E, 6 (15%) K666N/Q/R, 3 (8%) E622D, 2 (5%) H662D/Q, 2 (5%) Y623C, 1 (3%) R625C, and 1 (3%) T663I. One patient each displayed both SF3B1K700E and IDH2R140Q or SF3B1K700E and JAK2V617F. SF3B1 mutational frequencies were 69% for RARS, 36% for RCMD-RS, 18% for RAEB1-RS and 0% for RAEB2-RS (p=0.0007). SF3B1 mutations clustered with normal (59%) and other very good/good risk (57%) karyotype and were infrequent in poor/very poor karyotype (5%; p=0.0004). Additional significant correlations were noted between SF3B1 mutations and lower IPSS-R risk category (p=0.0004), advanced age (p=0.006), higher platelet count (p=0.0003). Results II: SF3B1 mutations and prognostic relevance in MDS-RS: To date, 68 (∼77%) deaths and 10 (∼11%) leukemic transformations have been documented. Median follow-up for living patients was 82 months. Univariate survival analysis considering all 88 study patients identified the following as strongly significant risk factors: IPSS-R (p<0.0001), WHO morphologic categories (p<0.0001), cytogenetic risk categories (p<0.0001), and transfusion need (p=0.0003). In multivariable analysis, only IPSS-R and transfusion need remained significant. In univariate analysis, the presence of SF3B1 mutations was significantly associated with better overall (p=0.04) and leukemia-free (p=0.03) survival; however, in both instances, significance was completely accounted for by WHO morphologic risk categorization and the morphology-adjusted p values were 0.6 and 0.2, respectively. In other words, when RARS and RCMD were analyzed separately, there was no additional prognostic value from the presence or absence of SF3B1 mutations (Figures 1 and 2; p=0.67 and 0.44, respectively). Conclusions: SF3B1 mutations are prevalent in RARS and RCMD-RS but do not provide additional, WHO morphology-independent or IPSS-R-independent, prognostic information. Disclosures: No relevant conflicts of interest to declare.

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Impaired Mitophagy of Nucleated Erythroid Cells Leads to Anemia in Patients with Myelodysplastic Syndromes

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/6328051 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

Huijuan Jiang ◽

Liyan Yang ◽

Lifang Guo ◽

Ningbo Cui ◽

Gaochao Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Myelodysplastic Syndromes ◽

Blood Cells ◽

Common Symptom ◽

Ineffective Erythropoiesis ◽

Nucleated Red Blood Cells ◽

Erythroid Precursors ◽

Ring Sideroblasts ◽

Novel Target

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by cytopenia and dysplasia. Anemia is the most common symptom in patients with MDS. Mitophagy and mitochondrial dysfunction might be involved in the development of MDS. In this study, we investigated the change of mitophagy in erythroid precursors in MDS patients. We found that NIX-mediated mitophagy was impaired in bone marrow nucleated red blood cells (NRBC) of MDS patients, associated with an increased amount of damaged mitochondria and increased ROS level which might lead to apoptosis and ineffective erythropoiesis. The results showed that the amount of mitochondria in GlycoA+NRBC positively correlated with the count of ring sideroblasts in bone marrow samples. Meanwhile, the level of autophagy-associated marker LC3B in GlycoA+NRBC had a positive correlation with hemoglobin (Hb) levels, and the amount of mitochondria in GlycoA+NRBC had a negative correlation with Hb levels in high-risk MDS patients. Our results indicated that mitophagy might involve the pathogenesis of anemia associated with MDS. Autophagy might be a novel target in treatments of MDS patients.

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