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Pancytopenia is a common hematological abnormality encountered in clinical practice. We here report a 36-year-old male who presented to emergency department with complaints of weakness of bilateral lower limbs, burning sensation in all four limbs with history of loose stools, and vomiting 5 days back. The complete blood count of patient showed pancytopenia with no circulating atypical cells. Bone marrow examination performed showed nonspecific but characteristic findings. After excluding the possibility of infective etiology, a possibility of heavy metal toxicity was suspected in multidisciplinary meeting. The urine and blood levels of arsenic done came out very high, and a diagnosis of arsenic poisoning was made. Patient had multisystemic involvement with features characteristic of arsenic poisoning. The present case was a diagnostic challenge in face of nonforthcoming history. This case beautifully highlighted the importance of multidepartmental approach in such cases to arrive at unerring diagnosis and the unique bone marrow findings, although nonspecific were sufficient enough to indicate the possibility of acute insult to the hematopoiesis. How to cite this article: Nair RR, Singh PK, Sharma J, Gambhir I, Khanna S, Jain AK, et al. An Unusual Case of Pancytopenia: The Lessons Learnt. Indian J Crit Care Med 2022;26(1):141–144.

BACKGROUND Cytopenias have been reported in dogs treated with phenobarbital, but detailed descriptions of bone marrow findings and response to treatment are lacking. OBJECTIVES We aimed to characterize the hematologic findings and clinical outcomes of dogs that had been receiving phenobarbital at the time of marrow evaluation. METHODS Archived bone marrow slides and clinicopathologic data were reviewed in dogs undergoing marrow evaluation for any hematologic problems that developed while receiving phenobarbital (2008-2020). Dogs were excluded if marrow samples lacked diagnostic value, phenobarbital was withdrawn >1 day before marrow collection, a same-day complete blood count (CBC) was lacking, or dogs had concurrent illness or therapy known to cause cytopenias. RESULTS Thirteen dogs met inclusion criteria: eight pancytopenic, three anemic/thrombocytopenic, one neutropenic/thrombocytopenic, and one nearly neutropenic. Neutropenia was marked (

Morphologic interpretation is the standard in diagnosing myelodysplastic syndrome (MDS), but it has limitations, such as varying reliability in pathologic evaluation and lack of integration with genetic data. Somatic events shape morphologic features, but the complexity of morphologic and genetic changes makes clear associations challenging. This article interrogates novel clinical subtypes of MDS using a machine-learning technique devised to identify patterns of cooccurrence among morphologic features and genomic events. We sequenced 1079 MDS patients and analyzed bone marrow morphologic alterations and other clinical features. A total of 1929 somatic mutations were identified. Five distinct morphologic profiles with unique clinical characteristics were defined. Seventy-seven percent of higher-risk patients clustered in profile 1. All lower-risk (LR) patients clustered into the remaining 4 profiles: profile 2 was characterized by pancytopenia, profile 3 by monocytosis, profile 4 by elevated megakaryocytes, and profile 5 by erythroid dysplasia. These profiles could also separate patients with different prognoses. LR MDS patients were classified into 8 genetic signatures (eg, signature A had TET2 mutations, signature B had both TET2 and SRSF2 mutations, and signature G had SF3B1 mutations), demonstrating association with specific morphologic profiles. Six morphologic profiles/genetic signature associations were confirmed in a separate analysis of an independent cohort. Our study demonstrates that nonrandom or even pathognomonic relationships between morphology and genotype to define clinical features can be identified. This is the first comprehensive implementation of machine-learning algorithms to elucidate potential intrinsic interdependencies among genetic lesions, morphologies, and clinical prognostic in attributes of MDS.

BACKGROUND: The hallmark of myelodysplastic syndrome (MDS) remains dysplasia in the bone marrow (BM). However, diagnosing MDS may be challenging and subject to inter-observer variability. Thus, there is an unmet need for novel objective, standardized and reproducible methods for evaluating dysplasia. Imaging flow cytometry (IFC) offers combined analyses of phenotypic and image-based morphometric parameters, for example, cell size and nuclearity. Hence, we hypothesized IFC to be a useful tool in MDS diagnostics.METHODS: Using a different-from-normal approach, we investigated dyserythropoiesis by quantifying morphometric features in a median of 5953 erythroblasts (range: 489-68,503) from 14 MDS patients, 11 healthy donors, 6 non-MDS controls with increased erythropoiesis, and 6 patients with cytopenia.RESULTS: First, we morphometrically confirmed normal erythroid maturation, as immunophenotypically defined erythroid precursors could be sequenced by significantly decreasing cell-, nuclear- and cytoplasm area. In MDS samples, we demonstrated cell size enlargement and increased fractions of macronormoblasts in late-stage erythroblasts (both p CONCLUSION: We demonstrate proof-of-concept results of the applicability of automated IFC-based techniques to study and quantify morphometric changes in dyserythropoietic BM cells. We propose that IFC holds great promise as a powerful and objective tool in the complex setting of MDS diagnostics with the potential for minimizing inter-observer variability.

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

Context: Bone marrow abnormalities reported in various stages of HIV infection have not attracted much attention; this is partly because the clinical picture of HIV infection is still dominated by opportunistic infections and malignancies. Aims: 1. To study the bone marrow abnormalities in patients with HIV / AIDS and to find their association with peripheral hematological abnormalities. 2. To draw conclusions and compare with previous studies.Settings and Design: The present Prospective study was conducted at the Department of pathology, Kamineni Institute of Medical Sciences, Narketpalli over a period of 2 years starting from January 2017 to Dec 2018. Methods and Material: The study population included 54 newly detected HIV positive symptomatic or asymptomatic patients. Patients were classified into two clinical groups according to NACO criteria 1) AIDS and 2) Non-AIDS. Bone marrow analysis was done for HIV patients who presented with anemia, leucopenia, Thrombocytopenia and Bi-cytopenia. Results: Patients were classified into two clinical groups: AIDS (55.5%) and NON-AIDS (44.5%). Most common age group involved was 21-40 years and Male to Female ratio is 4:1 in AIDS Group and 1.6:1 in Non-AIDS group. Most common haematological abnormality was Anaemia present in 73.3% of AIDS group and 79.1% of Non-AIDS group. Bone marrow was predominantly normocellular (AIDS: 50%, Non-AIDS: 62.5%) with normoblastic maturation. Myelodysplasia was predominantly seen in Granulocytic series (AIDS: 26.6%, Non-AIDS: 12.5%). Erythroid dysplasia was reported in 23.3% of cases of AIDS group and 8.3% in Non-AIDS group. Megakaryocytic dysplasia was seen in 2 cases of AIDS group. Myelodysplasia was common in patients with CD4 counts < 200/µL. Patients with myelodysplasia presented with anaemia (52.9%), Leukopenia (41.1%) and Thrombocytopenia (11.7%).Conclusions: Bone marrow examination is usually performed in HIV patients to evaluate peripheral cytopenia. Myelodysplasia commonly observed in HIV patients predominantly involves Granulocytic series. Erythroid dysplasia is prominent in AIDS Patients. Myelodysplasia associated with anemia carries poor prognosis and marks progression to advanced disease. Bone marrow abnormalities evolve and increase in frequency during progression of HIV disease and thorough schematic evaluation carries prognostic significance.

We and others recently described families with germline heterozygote mutations in ETV6 leading to autosomal dominant highly penetrant thrombocytopenia, red cell macrocytosis and predisposition to leukemia.The bone marrow of affected individuals shows erythroid dysplasia and hyperplasia of small, hypolobulated immature megakaryocytes suggesting a differentiation arrest. This discovery led to subsequent studies that confirmed our findings and to additional larger studies that demonstrated a 1% frequency of germline ETV6 mutations among 4405 individuals with acute lymphoblastic leukemia. Additionally, a 4.5% prevalence of ETV6 germline mutations was reported in families with inherited thrombocytopenia. Preliminary data suggest that decreased ETV6 function leads to MK maturation arrest, impaired platelet production and differentially expressed platelet transcripts among individuals affected with ETV6 mutations when compared to control relatives. Additionally, individuals with some ETV6 mutation exhibit bleeding that appears disproportionate to the mildly reduced platelet count, suggesting a platelet function deficit. Furthermore, recent studies describe decreased ability of platelets from individuals with ETV6 mutations to spread on fibrinogen covered surfaces. Overall, ETV6 germline mutations represent a new cancer predisposition thrombocytopenia with platelet dysfunction.

Background In patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)-- suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability. Methods Bone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo. Results Here, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR-2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain. Conclusions This data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS. Electronic supplementary material The online version of this article (10.1186/s40364-018-0149-4) contains supplementary material, which is available to authorized users.

Nd1-S is the nuclear-localizing short variant form of Nd1 (Ivns1abp) encoding a Kelch family transcription factor. While the function of Nd1 has been investigated in the context of metastasis and doxorubicin-induced cardiotoxicity, little is known about its role in hematopoiesis. In this study, we investigated the function of Nd1-S in hematopoiesis by transplanting the Nd1-S-overexpressing murine hematopoietic stem and progenitor cells (HSPCs) into recipient mice (Nd1-S mice). Enforced expression of Nd1-S led to erythroid and megakaryocyte dysplasia, demonstrated by dramatically decreased red blood cells and platelets, and megakaryocytes in the peripheral blood and bone marrow of the Nd1-S mice. Moreover, phenotypic megakaryocyte-erythroid progenitors (MEPs) accumulated in these Nd1-S mice with aberrant morphology and defective colony-forming capability. Furthermore, these phenotypic MEPs showed impaired pathways regulating erythroid differentiation and megakaryocyte development. Therefore, our study provides de novo evidence that overexpression of Nd1-S in HSPCs leads to erythroid and megakaryocyte dysplasia in vivo by targeting MEPs.

A healthy 14-year-old, male neutered, Miniature Poodle was found to have a persistent erythrocyte macrocytosis and reticulocytosis with a normal and stable HCT. The hematologic features of macrocytosis, increased Howell-Jolly bodies, and metarubricytosis, in the absence of anemia or other cytopenias, combined with the cytologic evidence of bone marrow erythroid dysplasia, including megaloblastosis, binuclearity, increased mitotic activity, and nuclear fragmentation, are consistent with previous reports of congenital dyserythropoiesis termed poodle macrocytosis. We speculate that the additional presence of persistent reticulocytosis in the absence of an identifiable stimulus for accelerated erythropoiesis may represent a phenotypic variation of this inherited condition, and the morphologic abnormalities of the dyserythropoiesis are described.

Glucose-6-phosphate isomerase (GPI) deficiency, a genetic disorder responsible for chronic nonspherocytic hemolytic anemia, is the second most common red blood cell glycolytic enzymopathy. We report three patients from two unrelated families of Czech and Slovak origin with macrocytic hemolytic anemia due to GPI deficiency. The first patient had 15% of residual GPI activity resulting from two new heterozygous missense mutations c.478T > C and c.1414C > T leading to substitutions p.(Ser160Pro) and p.(Arg472Cys). Two other patients (siblings) inherited the same c.1414C > T p.(Arg472Cys) mutation in a homozygous constitution and lost approximately 89% of their GPI activity. Erythroid hyperplasia with dysplastic features was observed in the bone marrow of all three patients. Low hepcidin/ferritin ratio and elevated soluble transferrin receptor detected in our GPI-deficient patients suggest disturbed balance between erythropoiesis and iron metabolism contributing to iron overload.

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre-mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2-mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2-mutated mice. RNA sequencing of the Srsf2-mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r, Fyn, Gnas, Nsd1, Hnrnpa2b1, and Trp53bp1 Among the mutant Srsf2-associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults.

Background: Cytopenias are common in systemic lupus erythematosus (SLE), and it is fundamental to determine their etiology in order to establish an adequate therapeutic strategy. Objectives: To describe the findings in the bone marrow aspirations (BMA) and biopsies of patients with SLE and cytopenias, as well as clinical and laboratory features associated with the etiology of the hematological abnormalities. Methods: We performed a retrospective study in a third-level hospital in Mexico City. We included patients who fulfilled ACR criteria for SLE, presented with cytopenias and had a BMA and biopsy performed between 2000 and 2016. We described the main aspirate and biopsy findings, and also analyzed the final diagnosis and its association with clinical, laboratory and serological features. Results: We included 101 patients; median age was 32 years and 81.2% were women. Leukopenia ( Regarding bone marrow findings, there was erythroid dysplasia in 50.5% of patients, granulocytic dysplasia in 28.7% and megakaryocytic dysplasia in 16%. Myelofibrosis was found in 2.6%. An increase in plasma cells (≥5%) was found in 21.8%. In 72.3% of patients, bone marrow interpretation was conclusive. The most common diagnoses were disease activity (24.8%) and drug-associated myelotoxicity (28.7%). When compared to other etiologies, in patients with cytopenias secondary to disease activity, it was more frequent for the bone marrow be hypercellular (56 vs 23%, p=0.006) and to have increased megakaryocytes (40 vs 17.4%, p=0.048). Conversely, granulocytic dysplasia was less common in this group of patients (17.4% vs 54.3%, p=0.036) We analyzed factors associated with both activity and toxicity as final diagnoses (Table 1). After multivariate analysis, a neutrophil count Conclusion: The main causes of cytopenias in our SLE patients were disease activity and bone marrow toxicity. Our findings suggest that when patients present with less than 1000 neutrophils/µl, it is unlikely for the cytopenias to be secondary to disease activity. Our study reinforces the diagnostic utility of BMA and biopsy, and the associations we described may assist clinicians to determine the etiology of cytopenias in SLE patients, in order to make appropriate therapeutic decisions. Disclosure of Interests: Ana Barrera-Vargas: None declared, Jonathan Campos-Guzman: None declared, Samuel Govea-Pelaez: None declared, Aldo Garcia-Ramos: None declared, Roberta Demichelis-Gomez: None declared, Christianne Bourlon: None declared, Javier Merayo-Chalico Speakers bureau: Pfizer, Jorge Alcocer-Varela: None declared

International audience; Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the SF3B1 splicing factor gene. Patients with MDS with SF3B1 mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for eryth-ropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative ERFE transcript in patients with MDS with the SF3B1 mutation. Induction of this ERFE transcript in primary SF3B1-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an SF3B1 gene mutation than in patients with SF3B1 wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with SF3B1-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant ERFE transcript that was restricted to SF3B1-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.

Myeloid neoplasms with ring sideroblasts are currently categorized within the myelodysplastic syndromes (MDS) or myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in the World Health Organization classification. Recent findings have identified that the presence of ring sideroblasts in these disorders has a unique molecular basis, i.e., the somatic mutation of SF3B1, a gene encoding a splicing factor. Mutations of SF3B1 occur in up to 90% of patients with refractory anaemia with unilineage dysplasia (RARS) and 70% of those with refractory cytopenia with multilineage dysplasia and ring sideroblasts or RARS associated with marked thrombocytosis. Experimental evidence has shown that mutant SF3B1 results in the abnormal splicing of several genes, primarily due to misrecognition of 3' splice sites. The resulting aberrant mRNAs undergo nonsense-mediated mRNA decay (NMD), resulting in haploinsufficiency of canonical transcripts and protein expression. In addition, it is also possible that NMD-insensitive aberrant transcripts are translated into proteins with altered function. Patients with MDS carrying the SF3B1 mutation show a homogeneous disease phenotype characterized by isolated erythroid dysplasia and mild dysplasia in granulocytic or megakaryocytic lineages, supporting the notion that the SF3B1 mutation identifies a distinct entity within MDS. The available evidence suggests that these findings may have relevant impact on the diagnosis, classification and management of patients with these neoplasms.

Growth differentiation factor-15 (GDF-15) is a member of TGF-β superfamily. Among hematopoietic cells, this factor is mainly produced by erythroid series and is recently considered a biomarker of ineffective erythropoiesis (IE). Whether IE induces enhanced GDF-15 expression or is prompted by it, has remained elusive. In this study we investigated how high levels of GDF-15 contribute to IE-associated erythroid dysplasia. We assessed mRNA levels of GDF-15 during erythroid maturation as well as in patients with IE using qRT-PCR. Later, the erythroid colony-forming capacity of GDF-15-treated hematopoietic stem cells (HSCs) was evaluated by CFC assay. Any effect of elevated levels of GDF-15 on erythroid maturation was ultimately examined by expression analysis of erythroid-associated transcription factors and flow cytometry analysis of CD235a expression. GDF-15 mRNA expression increased during erythroid differentiation and also in β-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Treating the cells with high GDF-15 concentration (50 ng/ml) resulted in an approximate 30% decline in the capacity of erythroid colony formation of HSCs and CD235a positive cells. Additionally, erythroid-specific transcription factors showed significant down-regulation in the early stages of erythroid differentiation. According to the expression level of GDF-15 and the role it plays in the erythroid system, high-levels of this factor could be an auto-modulatory mechanism to control the excessive production of erythroid cells.

Gelsolin, an actin-remodeling protein, is involved in cell motility, cytoskeletal remodeling, and cytokinesis and is abnormally expressed in many cancers. Recently, human recombinant plasma gelsolin protein (pGSN) was reported to have important roles in cell cycle and maturation of primary erythroblasts. However, the role of human plasma gelsolin in late stage erythroblasts prior to enucleation and putative clinical relevance in patients with myelodysplastic syndrome (MDS) and hemato-oncologic diseases have not been reported. Polychromatic and orthochromatic erythroblasts differentiated from human cord blood CD34+ cells, and human bone marrow (BM) cells derived from patients with MDS, were cultured in serum-free medium containing pGSN. Effects of pGSN on mitochondria, erythroid dysplasia, and enucleation were assessed in cellular and transcriptional levels. With pGSN treatment, terminal maturation at the stage of poly- and ortho-chromatic erythroblasts was enhanced, with higher numbers of orthochromatic erythroblasts and enucleated red blood cells (RBCs). pGSN also significantly decreased dysplastic features of cell morphology. Moreover, we found that patients with MDS with multi-lineage dysplasia or with excess blasts-1 showed significantly decreased expression of gelsolin mRNA (GSN) in their peripheral blood. When BM erythroblasts of MDS patients were cultured with pGSN, levels of mRNA transcripts related to terminal erythropoiesis and enucleation were markedly increased, with significantly decreased erythroid dysplasia. Moreover, pGSN treatment enhanced mitochondrial transmembrane potential that is unregulated in MDS and cultured cells. Our findings demonstrate a key role for plasma gelsolin in erythropoiesis and in gelsolin-depleted MDS patients, and raises the possibility that pGSN administration may promote erythropoiesis in erythroid dysplasia.

BACKGROUND Despite bone marrow (BM) immunophenotyping by flow cytometry has progressively been recognized as an important tool for the diagnosis of myelodysplastic syndromes (MDS), the sparse knowledge about normal erythroid maturation and the lack of markers for erythroid characterization is a major shortcoming. METHODS Here, we analyzed the expression of CD43 and CD49d, two markers included in the diagnostic panel for B-cell chronic lymphoproliferative disorders (B-CLPD), in the CD34+ compartment of normal BM and along the normal and dysplastic erythroid maturation. For this, 13 normal BM aspirates and 18 BM aspirates from MDS patients were studied by flow cytometry. RESULTS Normal BM presented a higher expression of CD43 and CD49d among CD34+ erythroid precursors, compared to CD34+ cells committed to the remaining hematopoietic cell lineages. CD43 expression progressively decreased along the normal erythroid maturation, whereas CD49d levels increased from Stage I to Stage II, were maintained in Stages II and III, and then decreased until the last stage of maturation. In MDS, the expression of CD43 and CD49d followed a similar pattern, but with decreased expression levels for both markers, observed in all erythroid maturation stages (P < 0.05). CONCLUSIONS Our results point to the usefulness of CD43 and CD49d, two markers commonly present in B-CLPD diagnosis panels, in the identification of dysplastic phenotypic features in the erythroid lineage. This allows a feasible and inexpensive way to identify patients who would benefit from a more extensive study to evaluate the presence of MDS, during the processing of suspected B-CLPD samples. © 2019 International Clinical Cytometry Society.

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia Immunophenotyping by multiparameter flow cytometry (FC) is a technique that has been used for the hematologic neoplasm diagnosis, classification, prognosis and therapy monitoring. In the minimum diagnostic criteria, FC analysis of bone marrow (BM) cells was suggested as a co-criterion for the diagnosis of Myelodysplastic Syndromes (MDS) by the 2016 World Health Organization (WHO) Classification of Myeloid Neoplasms and Acute Leukemia. The molecular and cytogenetic techniques sensitivity and specificity, especially fluorescence in situ hybridization (FISH), used in MDS diagnosis, can be improved by FC mediated cell sorting. Despite its several advantages, FC immunophenotyping still faces several limitations in the contribution to the MDS diagnosis, namely the reduced knowledge about the erythroid maturation in normal MO and the lack of erythroid characterization markers, which makes the study of associated dysplasia less sensitive. The diagnostic panel for B-cell Chronic Lymphoproliferative Disorder (B-CLPD) includes markers whose expression shown to be altered in the erythroid lineage of MDS cases, namely CD43 and CD49d.This work consisted of two major objectives. The first consisted on the separation of the cells from the monocytic, granulocytic (neutrophilic) an erythroid lineages and the (CD34+/-/CD117+) cells from the BM of 55 cases with suspected MDS (71 ± 12 years), corresponding to 21 women and 34 men, in order to increase the FISH technique sensitivity (32 processed cases) and to detect the incidence of the most frequent genetic alterations in this entity, thus contributing to the diagnosis and prognosis. Chromosomal abnormalities were detected in only 17 processed cases, namely del (20q), monosomy 7, del (7q), del (5q), (-Y), and trisomy 8.The second objective was to study the expression pattern of CD43 and CD49d markers, both included in the diagnostic panel of B-CLPD, in the normal BM CD34+ compartment and in the erythroid maturation, being the latter studied in the normal BM and in MDS diagnosed cases. For this, 31 samples of BM aspirates were evaluated, of which 13 samples from normal BM and 18 samples from patients diagnosed with MDS. The MDS group was divided into MDS I (which includes MDS cases with single lineage dysplasia (n=2) and with multilineage dysplasia (n=6)); MDS II (which includes MDS cases with excess blasts 1 (n=3) and excess blasts 2 (n=7). Also, among all MDS cases (n=18), a third group of MDS with erythroid dysplasia demonstrated by morphology (n=7) was defined.In the CD34+ compartment, the expression of CD43 and CD49d shown to be much higher in the hematopoietic precursors compromised with the erythroid lineage compared to the precursors involved with the other lineages. In normal erythroid maturation, there was a decrease in the expression of CD43 across the different stages, with a high expression in stage I and a consequent decrease until the last stage. The expression pattern of CD49d was characterized by an increase in expression levels from stage I to stage II, by maintained expression between stage II and III and by a constant decrease from stage III to the last stage, which presented the lowest expression of this marker. In the MDS group, the expression of CD43 and CD49d showed to follow the same pattern observed in normal BM, however, a statistically significant decrease in mean fluorescence intensity (MFI) values was observed in all maturation stages for both markers, compared to the control group, particularly in MDS II and MDS with morphologic erythroid dysplasia.The results obtained during this study reinforce the contribution of FC in the MDS diagnosis and point to the usefulness of CD43 and CD49d markers in the erythroid maturation characterization, thus contributing to expand the panel of markers used for the study of this lineage, and since both markers are included in the B-CLPD diagnostic panel, could contribute to the concomitant diagnosis of a chronic B cell disorder and a possible MDS. A imunofenotipagem por Citometria de Fluxo multiparamétrica (CF) é uma técnica que tem sido utilizada para o diagnóstico, classificação, prognóstico e monitorização da terapêutica de neoplasias hematológicas. Nos critérios mínimos de diagnóstico, a análise de células da medula óssea (MO) por CF foi sugerida como um co-critério para o diagnóstico de Síndromes Mielodisplásicas (SMD) pela Classificação da Organização Mundial de Saúde (OMS) de Neoplasias Mieloides e Leucemia Aguda de 2016. A sensibilidade e especificidade das técnicas moleculares e citogenéticas, principalmente a técnica de hibridação in situ por fluorescência (FISH), utilizada no diagnóstico da SMD, podem ser melhoradas através da separação celular por CF. Apesar das suas várias vantagens, a imunofenotipagem por CF ainda enfrenta várias limitações ao nível da contribuição para o diagnóstico das SMD, nomeadamente o reduzido conhecimento sobre a maturação da linha eritroide, na MO normal e a falta de marcadores para a caracterização desta mesma linha, o que torna o estudo de displasias associadas pouco sensível. O painel de diagnóstico de Doenças Linfoproliferativas Crónicas de células B (DLPC-B) inclui marcadores cuja expressão mostrou ser alterada na linha eritroide em casos de SMD, nomeadamente o CD43 e o CD49d.Este trabalho engloba dois grandes objetivos. O primeiro consistiu na separação das células das linhas granulocítica (neutrófilo), monocítica e eritroide, e das células (CD34+/-/CD117+) da MO de 55 casos com suspeita de SMD (71 ± 12 anos), correspondendo a 21 mulheres e 34 homens, de forma a aumentar a sensibilidade da técnica de FISH (32 casos processados) e detetar a incidência das alterações genéticas mais frequentes nesta entidade, contribuindo desta forma para o diagnóstico e prognóstico. Foram detetadas anormalidades cromossómicas em apenas 17 dos casos processados, nomeadamente a del (20 q), monossomia do 7, del (7q), del (5q), anomalia (-Y), e a trissomia do 8.O segundo objetivo consistiu no estudo do padrão de expressão dos marcadores CD43 e CD49d no compartimento CD34+ da MO normal e na linha eritroide, sendo esta última estudada na MO normal e em casos diagnosticados com SMD. Para isso, foram analisadas 31 amostras de aspirados medulares referentes a 13 amostras de MO normal e 18 amostras de doentes com diagnóstico de SMD. O grupo SMD foi dividido em SMD I (que inclui casos de SMD com displasia unilinha (n=2) e casos com displasia multilinha (n=6)); SMD II (que inclui casos de SMD com excesso de blastos do tipo 1 (n=3) e tipo 2 (n=7)). Ainda, tendo em conta todos os casos de SMD (n=18), foi definido um terceiro grupo de SMD onde se observou displasia eritroide por morfologia (n=7). No compartimento CD34+ normal, a expressão de CD43 e CD49d mostrou ser bastante mais elevada nos percursores hematopoiéticos comprometidos à linha eritroide em comparação com os percursores comprometidos às outras linhas hematopoiéticas. Na linha eritroide normal, observou-se uma diminuição da expressão de CD43 ao longo da maturação, com uma elevada expressão no estádio I e com uma consequente diminuição até ao último estádio. O padrão de expressão de CD49d foi caracterizado por um aumento de expressão do estádio I para o estádio II, por uma expressão mantida entre o estádio II e III e por uma diminuição constante do estádio III até ao último estádio, que apresentou a menor expressão deste marcador. No grupo SMD, a expressão de CD43 e CD49d mostrou seguir o mesmo padrão observado na MO normal, no entanto, observou-se uma diminuição estatisticamente significativa nos valores da média da intensidade de fluorescência (MIF), em todos os estádios maturativos, para ambos os marcadores, em comparação ao grupo de controlo. Esta diminuição mostrou ser mais acentuada, e com significado estatístico, nos grupos SMD II e SMD com displasia eritroide, quando comparado com o grupo controlo, para ambos os marcadores. Os resultados obtidos ao longo do trabalho reforçam o contributo da CF no diagnóstico das SMD e apontam para a utilidade dos marcadores CD43 e CD49d na caracterização da maturação eritroide, contribuindo deste modo para expandir o painel de marcadores utilizados para o estudo desta linha, e uma vez que ambos os marcadores estão incluídos no painel de diagnóstico DLPC-B, poderiam contribuir para o diagnóstico concomitante de uma neoplasia crónica de células B e uma possível SMD.

International audience; An evaluation of the significance of specified dyserythropoietic features in suspected myelodysplastic syndrome (MDS) and acute myeloid leukaemia with erythroid dysplasia was made by means of evaluation of 100 electronic images of bone marrow erythroblasts from each of 20 subjects 11 with a myeloid neoplasm, six with another condition that could cause erythroid dysplasia and three healthy controls. The evaluation was carried out independently by seven experienced haematologists/haematopathologists who were blinded to the diagnosis. The majority of the dyserythropoietic features listed in the World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues were validated, although karyorrhexis was found to be infrequent and lacking in specificity; multinuclearity and megaloblastosis were more often observed but also lacked specificity. Good majority agreement on the identification of dysplastic features was obtained. Despite this, it was demonstrated that a reliable diagnosis of MDS can often not be made on the basis of erythroid morphology alone. Interpretation of dyserythropoiesis must be carried out with full knowledge of other clinicopathological features and with a constant awareness of the other conditions that can be confused with MDS. An iron stain is essential, as cases with ring sideroblasts may otherwise not be recognised as having MDS. © 2018 John Wiley and Sons Ltd

Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated erythroid dysplasia and 15% or more bone marrow ring sideroblasts. Ring sideroblasts are found also in other MDS subtypes, such as refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS). A high prevalence of somatic mutations of SF3B1 was reported in these conditions. To identify mutation patterns that affect disease phenotype and clinical outcome, we performed a comprehensive mutation analysis in 293 patients with myeloid neoplasm and 1% or more ring sideroblasts. SF3B1 mutations were detected in 129 of 159 cases (81%) of RARS or RCMD-RS. Among other patients with ring sideroblasts, lower prevalence of SF3B1 mutations and higher prevalence of mutations in other splicing factor genes were observed (P < .001). In multivariable analyses, patients with SF3B1 mutations showed significantly better overall survival (hazard ratio [HR], .37; P = .003) and lower cumulative incidence of disease progression (HR = 0.31; P = .018) compared with SF3B1-unmutated cases. The independent prognostic value of SF3B1 mutation was retained in MDS without excess blasts, as well as in sideroblastic categories (RARS and RCMD-RS). Among SF3B1-mutated patients, coexisting mutations in DNA methylation genes were associated with multilineage dysplasia (P = .015) but had no effect on clinical outcome. TP53 mutations were frequently detected in patients without SF3B1 mutation, and were associated with poor outcome. Thus, SF3B1 mutation identifies a distinct MDS subtype that is unlikely to develop detrimental subclonal mutations and is characterized by indolent clinical course and favorable outcome.

Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) is a provisional entity within myelodysplastic/myeloproliferative neoplasm (MDS/ MPN), unclassifiable in the 2008 World Health Organiza tion classification. This category includes patients that at the time of initial presentation manifest some diagnostic features (cytopenias and dysplasia) of MDS and others (overproduction of one or more myeloid cell lineages) of MPN. This dual clinical nature limits the distinct clas sification as either MDS or MPN. The diagnostic criteria include peripheral blood (PB; anemia, absence of blasts, platelets  450  10 9 /L) and bone marrow (BM) findings (erythroid dysplasia,  15% of erythroid precursors with RS,  5% blasts, and megakaryocytic hyperplasia with large atypical megakaryocytes) [1,2]. The value of RARS-T as a diagnostic category has remained in question over the years. There is a persistent debate on whether RARS-T is a disease evolving from MDS, is part of the MPN (an essential thrombocythemia (ET) that acquires RS) or is a result of two distinct disorders occurring simultaneously [3]. Data on survival outcomes have also been discordant. RARS-T patients have usually worse outcomes compared to MPN patients and are more likely to develop leukemia although RARS-T patients harboring JAK2 [4,5] or SF3B1 mutations appear to have better survival than wild-type RARS-T patients [5,6]. The presence of DNMT3A and ASXL1 mutations is noted in 17% and 10% of cases and confers an unfavorable prognosis [6,7]. In recent years, molecular analysis has unraveled an overlapping mutational spectrum of RARS-T with MPN or MDS. With the exception of SF3B1 mutations, which have been associated to the RS phenotype in RARS and RARS-T patients [6,8] and linked to the development of erythroid dysplasia, macrocytic anemia, splenomegaly and thrombocytosis in an SF3B1 /2 mouse model [9], the vast majority of mutational events and frequencies have not explained either the MPN or other features of the disease. Indeed mutations in JAK2, MPL, and TET2 are common (60%, 5–20%, and 26%

The t(8;21)(q22;q22) translocation is specifically observed in acute myeloid leukemia (AML) M2 subtype, whereas del(5q) is one of the most common cytogenetic aberrations in myelodysplastic syndromes (MDS). Thus, t(8;21)(q22;q22) and del(5q) appear to be mutually exclusive, and the association between them has not been characterized yet. Here, we report an 81-year-old woman with coexistent t(8;21)(q22;q22) and del(5q) at initial diagnosis. The bone marrow was infiltrated with 18.4% myeloblasts, and showed marked myeloid and erythroid dysplasia. Myeloblasts were positive for CD19 and CD56 as well as CD13, CD33, CD34 and HLA-DR. G-banding and spectral karyotyping showed 46,XX,del(5)(q?),t(8;21)(q22;q22)[18]/46,XX[2]. Both del(5)(q?) and t(8;21)(q22;q22) were present in a single clone. Fluorescence in situ hybridization (FISH) on metaphase spreads detected a RUNX1/RUNX1T1 fusion signal on the der(8)t(8;21)(q22;q22), and confirmed deletion of CSF1R signaling at 5q33-q34 on the del(5)(q?). Furthermore, FISH on interphase nuclei revealed that the RUNX1/RUNX1T1 fusion signal and deletion of CSF1R signaling were found in 66.0% and 58.0% of interphase cells, respectively, suggesting that del(5)(q?) occurred in cells with RUNX1/RUNX1T1. These results indicated a diagnosis of AML with t(8;21)(q22;q22)/RUNX1/RUNX1T1 rather than MDS, even though the percentage of bone marrow myeloblasts was less than 20%. Based on these findings, together with those of other reported cases, del(5q) seems to be an extremely rare but recurrent secondary aberration in AML with t(8;21)(q22;q22).

Background The only potentially curative therapy for myelodysplastic syndrome is allogeneic hematopoietic cell transplant; unfortunately, there is a high relapse rate. The objective of this study was to perform a detailed clinicopathologic study of patients with relapsed myeloid neoplasm following allogeneic hematopoietic cell transplant for myelodysplastic syndrome. Methods Pre-transplant, post-transplant, and relapse bone marrow and peripheral blood morphologic features (including dysplasia) were retrospectively evaluated by study authors. Clinical features and results of cytogenetic analysis and engraftment/chimerism studies were obtained from the medical record. Results Our study describes 21 patients with a median time to relapse of 6 months (range 2–82). Ten of the patients relapsed with higher grade disease, including six with overt acute myeloid leukemia. Pre-transplant megakaryocyte dysplasia was associated with dysplastic megakaryocytes in the relapse specimen; however, neither erythroid dysplasia nor granulocytic dysplasia were associated with their counterpart in the relapse specimen. Relapse specimens had a lower marrow cellularity and higher blast percentage than pre-transplant disease. Cytogenetic comparisons before and after transplant showed variety, including clonal evolution (22%), the same abnormal clone (33%), or a different abnormal clone (22%). Conclusions Our detailed review of post-transplant marrow biopsies prior to relapse highlights the difficulty in diagnosing relapse and particularly impending relapse.

Mutations (MT) in the 5' untranslated region (UTR) of ANKRD26 (A26) are implicated in ANKRD26- related thrombocytopenia (A26-RT), an autosomal dominant disorder of mild to moderate thrombocytopenia (TP) often presenting in adulthood, although severe and pediatric cases are reported. Erythrocyte and leukocyte counts are normal to increased, with unremarkable morphology. Platelet (plt) size is usually normal, as with ETV6- and RUNX1-mutated TP. Together, A26, ETV6, and RUNX1 germline (GL) MT comprise a separate 2016 WHO category of myeloid neoplasms (MN) with GL predisposition and preexisting platelet disorders. Normal plt size separates A26-RT from other familial TPs with giant plts. No consistent morphological plt aberrations have been reported. Bleeding history is absent or mild, and while TP is not life threating, 8-10% of patients (pts) develop a MN, including a 30-fold increased risk for AML relative to the general population. A26 is an inner membrane adaptor protein with 2 major domains: ankyrin repeats (ANKR) and coil-coil (C-C), both of which interact with signaling and cytoskeletal proteins. A26-RT MT are almost exclusively in the 5'UTR, however, rare A26 coding variants (A26-CV) are reported to segregate with familial TP. Still, some studies on A26-RT limit sequencing to the 5'UTR. As such, A26-CV are not well represented or described. We performed whole-exome sequencing (WES) on 195 pts with MN seen at Cleveland Clinic between 2004 and 2012, and downloaded WES .bam files from online sources, totaling 653 MN cases. Using a standard pipeline for discovery of rare GL variants ( Complete clinical description was available for 8 A26-CV pts, all diagnosed with MDS or MDS/MPN at med age of 64. Two pts had antecedent bruising 1 year prior to diagnosis (dx). No prior bleeding was noted. Three pts had prior TP, two of whom had bicytopenia and pancytopenia. First degree family history (FH) was positive for cancer in 6 pts (75%), including 2 pts with FH of hematologic neoplasms. One pt had a 2nd-degree relative with a non-malignant hematologic condition requiring transfusions. At dx, cytogenetics were normal and complex in 2 pts each (25%). Deletions in chromosomes 5, 17, 20, and Y were observed in 1 pt each, and monosomy 7 in 2 pts. On bone marrow aspirate, 6 pts (75%) had dysmegakaryopoiesis, found in interstitial patterns and clusters. Mild to moderate dyserythropoiesis was observed in 5 pts (63%), and 5 pts had dysgranulopoiesis. The med blast percentage was 1.5% (range 0-8%), with 5 pts having hypercellular marrow, med 65% (range 40-95%). On corresponding CBC, pts were anemic (med hemoglobin of 9.5 g/dL), with the majority (n=5) showing signs of erythroid dysplasia, and also thrombocytopenic (med plt of 99 k/μL), with giant plts observed in 3 pts. Two pts had both giant plts and abnormal erythroid morphology. Hypo and monolobate, hyposegmented, and hypogranular forms were observed in the 3 lineages, as well as detached nuclear lobes, budding, segmentation, and nuclear-cytoplasmic dyssynchrony. On next generation sequencing, co-occurring SRSF2 and ASXL1 MT were observed in 3 and 2 pts, respectively. In sum, we have identified 22 A26-CV in MN, suggesting a role in predisposition as with A26-RT. We have seen in our cohort that A26-CV pts present differently from those with A26-RT. They have a variable past medical history and limited FH of TP, are anemic, with multilineage dysplasia observed not just in bone marrow, but also on peripheral blood smear, especially in megakaryocytes and erythrocytes. This is not surprising, as A26 is expressed in both lineages. The presence of giant plts is noteworthy. The mechanism for hypomorphic A26-CV may differ from that of the A26 5' UTR, which increase A26 levels in late-stage megakaryopoiesis by abrogating RUNX1/FLI1 binding, leading to aberrant proplatelet formation. Given the plt size and presence of nuclear phenotypes, altered interactions with signaling and cytoskeletal proteins could be involved, and may represent a novel A26 phenotype. Further investigation and association of A26-CV with MN ontogeny is under way. Disclosures Mukherjee: Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau. Advani:Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Research Funding. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Gerds:Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Roche: Research Funding; Incyte: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

Del (9q) is a recurrent cytogenetic abnormality in acute myeloid leukaemia (AML). We report an analysis of 81 patients with del(9q) as a diagnostic karyotypic abnormality entered into the Medical Research Council AML trials 10, 11 and 12. Patients were divided into three groups: (i) Sole del (9q), 21 patients ; (ii) Del(9q) in association with t(8 ; 21), 29 patients ; (iii) Del(9q) in association with other cytogenetic abnormalities, 31 patients. Sole del(9q) was associated with a characteristic bone marrow phenotype at diagnosis: a single Auer rod was found in all cases examined. There was also an association with erythroid dysplasia (74%) and granylocytic lineage vacuolation (90%). The incidence of all three of these features was significantly higher (P < 0.05) in the sole del(9q) group compared with control cases lacking del(9q). The overall survival (OS) of all 81 patients was compared with a control group of 1738 patients with normal cytogenetics entered in the same trials over the period of investigation. The 5-year OS for patients with del(9q) was 45%, compared with 35% for the control group (P = 0.09). Patients with del(9q) in association with t(8 ; 21) had a 5-year OS of 75%, which was significantly better than the groups with either sole del(9q) (40%) and del(9q) with other abnormalities (26% ; P = 0.008). Karyotyping indicated a common area of deletion in the region 9q21-22, which was present in 94% of cases. It is likely that the deletion of single or multiple tumour suppressor genes located in this region may underlie the pathogenesis of del (9q) AML.

Abstract 924 Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders that are commonly characterized by anemia due to ineffective hematopoiesis. Even though a third of patients with MDS may transform to acute leukemias, cytopenias drive morbidity for most patients. Anemia remains a major cause of morbidity from fatigue. Most of the morbidity experienced by such patients is due to low red blood counts and therefore studies on the molecular pathogenesis of dysplastic erythropoiesis leading to anemia are critically needed. We have identified DOCK4, an important cofactor for various GTPases located on the chromosome 7q segment as a novel silenced gene in MDS and show that it's down regulation leads to disruption of normal erythropoiesis. In an attempt to uncover genes aberrantly expressed in MDS, we initially performed an integrative genomic analysis of primary hematopoietic cells from MDS patients. These studies revealed that DOCK4 is significantly under-expressed and hypermethylated in MDS stem and progenitor cells. Immunohistochemcial analysis revealed significantly reduced levels of DOCK4 in MDS erythroblasts. We then evaluated DOCK4 expression in a large published cohort of MDS gene expression datasets (N=183) and found that DOCK4 expression was strikingly reduced in the subset of 55 MDS patients with refractory anemia (RA; P value = 0.006). The RA subset of patients only has isolated anemia as the clinical presentation and has no apparent abnormalities in white cells or platelets. This association strongly alludes to a role of DOCK4 down-regulation in the erythroid dysplasia and anemia seen in this disease. Inorder to elucidate the functional implications of aberrant DOCK4 expression during erythropoiesis we used a dynamic model of human erythropoiesis to determine normal expression pattern during terminal differentiation and the impact of silencing DOCK4 expression on healthy primary erythroblasts. These studies revealed that DOCK4 is highly expressed during late stages of normal erythropoiesis and knockdown of DOCK4 in primary erythroblasts disrupted the F-actin skeleton. We then examined F-actin skeletal disruption in CD34+ derived erythroblasts from MDS patients. In order to quantify the extent of actin filament disruption directly in patient derived erythroblasts we first developed an assay based on multispectral flow cytometry (ImageStream™). This assay not only allowed us to visualize individually F-actin-stained cells but also allowed us to determine the percentages of cells in a given sample that contained shorter fragmented F-actin. These experiments revealed that approximately 85% of the cells in MDS patients with -7q deletion and/or hypermethylated promoter region in the DOCK4 gene contained shorter disrupted actin compared to the healthy controls that showed only 10% of the cells with disrupted F-actin. The level of F-actin disruption in healthy samples treated with cytochalasin D, an inhibitor of actin polymerization was 90%. We then examined the membrane stability of -7q MDS erythrocytes by performing osmotic fragility assays and found that these patients possessed erythrocytes that were more fragile compared to healthy erythrocytes. Based on these results we conclude that DOCK4 is an important signaling intermediate that is instrumental in maintaining erythroblast membrane homeostasis and silencing of DOCK4 in MDS contributes to anemia. Disclosures: No relevant conflicts of interest to declare.

Myelodysplastic syndromes (MDS) are clonal disorders of haematopoiesis characterised by dysplastic changes of major myeloid cell lines. However, the mechanisms underlying these dysplastic changes are poorly understood. Here, we used a genetically modified mouse model and human patient data to examine the physiological roles of H2AX in haematopoiesis and how the loss of H2AX contributes to dyserythropoiesis in MDS. H2AX knockout mice showed cell-autonomous anaemia and erythroid dysplasia, mimicking dyserythropoiesis in MDS. Also, dyserythropoiesis was increased in MDS patients with the deletion of chromosome 11q23, where H2AX is located. Although loss of H2AX did not affect the early stage of terminal erythropoiesis, enucleation was decreased. H2AX deficiency also led to the loss of quiescence of hematopoietic stem and progenitor cells, which dramatically compromised their bone marrow engraftment. These results reveal important roles of H2AX in late-stage terminal erythropoiesis and hematopoietic stem cell function.

Nuclear transcription factor Stat3 is important for proper regulation of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) proliferation, survival, and cytokine signaling responses. A new, noncanonical role for Stat3 in mitochondrial function has been discovered recently. However, there is little information on the role(s) of mitochondrial Stat3 in HSC/HPC function, especially potential effects of Stat3/mitochondrial dysregulation in human diseases. We investigated hematopoietic cell–targeted deletion of the STAT3 gene in HSCs/HPCs with a focus on mitochondrial function. We found that STAT3−/− mice, which have a very shortened lifespan, dysfunctional/dysregulated mitochondrial function and excessive reactive oxygen species production in HSCs/HPCs that coincides with pronounced defects in function. These animals have a blood phenotype with similarities to premature aging and to human diseases of myelodysplastic syndrome and myeloproliferative neoplasms such as erythroid dysplasia, anemia, excessive myeloproliferation, and lymphomyeloid ratio shifts. We show herein that the lifespan of STAT3−/− animals is lengthened by treatment with a reactive oxygen species scavenger, which lessened the severity of the blood phenotype. These data suggest a need for more detailed studies of role(s) of Stat3 in HSC/HPC mitochondrial function in human diseases and raise the idea that mitochondrial Stat3 could be used as a potential therapeutic target.

Background/Aim: Abnormal immunophenotypes of haematopoietic cells in myelodysplastic syndromes (MDS) have been identified by flow cytometry (FCM) as a typical characteristic of myeloid dysplasia. Considering that most MDS patients show varying degrees of erythroid dysplasia, we analysed the immunophenotypic feature of erythroblasts to evaluate its diagnostic application in MDS. Methods: Erythroid antigens CD71 and CD105 expression were analysed using FCM. The development index (DI) acquired by log transformation of the CD71/CD105 expression ratio was used to denote the erythroid maturation and distinguish patients with non-clonal cytopenias from low-risk MDS. The diagnostic quality of DI in distinguishing MDS from non-clonal cytopenia patients was evaluated using the receiver–operator characteristic (ROC) curve. Results: Under-expression of CD71 and over-expression of CD105 were detected in erythroblasts of MDS patients compared with non-clonal cytopenias. The diagnostic test showed good diagnostic power that the area under the ROC curve was greater than 0.9. The diagnostic sensitivity and specificity were 75.6% and 92.3%, respectively, according to the DI threshold defined by the ROC curve in low-risk MDS patients with normal karyotypes. Moreover, the DI showed a positive correlation with the haemoglobin level, and the MDS patients with lower DI usually showed frequent red cell transfusion. The patients with a lower DI generally had the HLA-DR15 allele or marrow hypocellularity. Conclusion: Developmental defects and immune-associated factors may contribute to the erythroid dysplasia. The DI derived from ratios of CD71 and CD105 expression is a useful marker to characterise dyserythropioiesis associated with MDS and can be helpful in distinguishing it from dyerythropoiesis associated with non-clonal disorders.

Introduction: The IMDSFlow working group reported recently the usefulness of immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes (MDS) (Westers, 2017). This multicenter study revealed that analysis of CD36 and CD71 expression on nucleated erythroid cells as coefficient of variation (CV), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between MDS and non-clonal cytopenias. These four parameters were analyzed on bone marrow samples after red blood cell lysis (RBC) procedure. Since this latter could also remove some nucleated erythroid cells, it was proposed to use a nuclear dye in a whole no-lysis bone marrow strategy for analysis of CD36 and CD71 expression (Mathis, 2013). So far, no study has compared the four ELN dyserythropoiesis parameters after or without lysis procedure. Objective:We aimed to evaluate whether no-lysis procedure can lead to a better assessment of dyserythropoiesis by flow cytometry compared to lysis procedure by preserving erythroblast cells. Methods: One hundred patients referred to our laboratory for bone marrow investigations between November 2017 and June 2018 were included in this prospective study. Nineteen patients were used as control samples without cytopenia whereas 81 presented at least one cytopenia. Complete blood count parameters as well as morphological and cytogenetic analyses were used to classify these patients as 25 MDS or 56 non-MDS, referred thereafter as pathological controls. Bone marrow specimens were collected on EDTA and were processed within 4h following aspiration. A similar amount of each sample was stained in parallel with the same antibody panel (CD36, CD71, CD117 and CD45), yet according to two different protocols, one with CyTrak orange without lysis procedure and one with RBC lysis using VersaLyse (Beckman-Coulter). Data were acquired using a Navios cytometer and analyzed using the Kaluza software (BC). Geometrical means of fluorescence (GMFI) of CD71 as well as CD36 and CD71 coefficients of variation and the percentage of CD117+ erythroid progenitors were collected in order to calculate the ELN dyserythropoiesis score as previously described (see Figure 1 A-B for erythroblast selection). Results: Firstly, we compared the percentages of erythroblasts obtained with the no-lysis and the RBC lysis strategies as reported to the total nucleated cells analyzed (corresponding to the CD45 positive cells in addition to erythroblasts selected as CD71 and CD36 positive cells). Surprisingly, we found that the lysis protocol led to a higher percentage of erythroblasts than the no-lysis protocol (19.0±11.8% vs 15.4±10.8%; p Samples stained according to the two different protocols were analyzed with the same calibrated cytometer and the same settings for all markers (especially CD71 and CD36), allowing comparison of the raw data. The four parameters of the ELN score were significantly different between the two protocols for the 100 patients analyzed: both CV of CD36 and CD71 and % CD117 progenitors were higher with no-lysis protocol while GMFI CD71 was lower (Figure 1 C-F). We then calculated the ELN score with the recommended weighted manner: 4 points for increase in CD36 CV, 3 points for increase in CD71 CV, 2 points for decreased CD71 MFI and 2 points in the case of decreased or increased percentage of CD117+ erythroid cells. For each of the four parameters, cut-offs for the identification of MDS-associated aberrancies were defined against the 10th and/or 90th percentiles of the values obtained either from control samples (Figure 1 G) or from pathological controls (Figure 1 H) for both lysis and no-lysis protocols. With cut-offs defined against control samples, both specificity and sensitivity were better for lysis vs no-lysis protocol (93.3% vs 88.0% and 20.0% vs 12.0%, respectively). With cut-offs defined against pathological controls, again both specificity and sensitivity were higher for lysis vs no-lysis protocol (94.7% vs 88.0% and 20.0% vs 8.0%, respectively). Conclusion: Our results demonstrate that the lysis protocol leads to better specificity and sensitivity of the ELN dyserythropoiesis score than the no-lysis protocol with cut-offs defined against either control samples or pathological controls. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Morphology has dominated the diagnosis and classification of MDS for decades. With the advent of NGS, morphology is used as a gold standard to assess associations of specific disease sub-entities with individual mutations or their combinations. An example is the association between SF3B1 mutations and ringed sideroblasts, or with disease-defining lesions, such as t(8:21), 11q23 translocations, and EVI1 mutations. Beyond these discrete examples, assignments of diverse morphologic features to combinations of specific genotypes has been more challenging. To identify potentially novel paradigms of associations between molecular hits and morphologies we examined 835 patients with MDS. Common somatic mutations, chromosomal abnormalities and morphologic reviews were subjected to blinded, objective standard diagnostic criteria. Abnormalities included individual lineage dysplasia and cytopenias, monocytosis, fibrosis, and increased megakaryocytes, all classified using standard criteria. To correlate the presence of mutations to morphologic criteria, only mutations with a clonal burden >10% were used. In total, 794 out of 835 patients (95%) harbored at least one lineage dysplastic feature. Myeloid, erythroid and megakaryocytic dysplasia occurred in 56% (470/835), 71% (590/835) and, 70% (587/835) of patients, respectively. Bi- and tri-lineage dysplasia were also identified in 36% (297/835) and 33% (278/835). In 90% (753/835) of patients there was cytopenia: 48% (402/835) had neutropenia, 61% (510/835) anemia, and 61% (510/835) thrombocytopenia. More than 50% of patients had multiple-cytopenias; 23% (194/835) had pancytopenia. Monocytosis was present in 20% (168/835) of patients and 31% (256/835) had elevated numbers of megakaryocytes. For analysis all possible combinations (25 pairs in total) were queried and those present in >80 patients were evaluated. Based on NGS among 36 genes, TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%),, were frequently mutated genes/CNAs, each present in >10% of patients. A number of relationships between phenotypic features and molecular context were uncovered. For example, patients with thrombocytopenia also commonly had tri-lineage dysplasia [38% (198/510) vs. 25%, (80/325), p When we performed statistical analyses that sought correlations between clinical features and genetic alterations, 78 significant associations were identified. For instance, patients with erythroid dysplasia more frequently had SF3B1 mutations [in 15% of erythroid dysplasia patients, 90/590] than patients without it [6%, 15/245] (p=.0002, q=.001). In contrast, these patients were less likely to have EZH2 mutations [4.2% (25/590) vs. 11.4% (28/245), p Using various conventional and more advanced strategies, we present the first comprehensive genotype-phenotype association study that linked somatic molecular events to morphologic and clinical features. We demonstrate that the complexities of how molecular context governs morphologic changes can be deciphered. Certain rigorously defined morphologic configurations show specific associations with individual mutations or their combinations. Disclosures Nazha: MEI: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Myelodysplastic syndromes (MDS) constitute a form of blood cancer that primarily affects the elderly and is characterized by anemia (or other cytopenias) and a high risk of leukemic transformation.1 The World Health Organization (WHO) system has formally classified MDS as 1 of 5 myeloid malignancies that also include acute myeloid leukemia (AML) and myeloproliferative neoplasms.2 All myeloid malignancies, including MDS, are clonal stem cell diseases. Unlike the case with BCR-ABL1-positive chronic myelogenous leukemia,3 the disease-causing mutation in MDS is currently unknown. It is generally believed that the MDS phenotype is additionally affected by secondary mutations, genetic haploinsufficiency, epigenetic changes, and altered host response that result in cytokine, immune, and bone marrow stromal changes.1 Unfortunately, none of this information has successfully been translated into a robust pathogenetic framework or effective targeted therapy. In routine clinical practice, MDS are suspected when an otherwise unexplained anemia is associated with other cytopenias, increased mean corpuscular volume, or increased red cell distribution width. The peripheral blood smear in MDS shows oval macrocytes, a dimorphic red blood cell population, or hyposegmented/hypogranulated neutrophils. Diagnosis requires bone marrow examination and cytogenetic studies. The consensual minimum criterion for diagnosis is the presence of erythroid, granulocyte, or megakaryocyte dysplasia in 10% or more of informative cells.2 In this regard, one must exclude the possibility of erythroid dysplasia associated with vitamin B12/folate/copper deficiency, viral infections, chemotherapy, or lead/arsenic poisoning. The detection of an abnormal karyotype confirms the morphologic diagnosis of MDS.4 The WHO system further subclassifies MDS into 6 subcategories depending on the percentage of blasts in the bone marrow or peripheral blood, presence or absence of bone marrow multilineage dysplasia, or excess ring sideroblasts: refractory cytopenia with unilineage dysplasia, refractory anemia with ring sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess of blasts, MDS associated with isolated 5q–, and MDS unclassifiable.2 Myelodysplastic syndromes are rare in young people, with an estimated incidence rate of less than 1 per 100,000 before age 50 years and approximately 2 per 100,000 between ages 50 and 60 years; however, the incidence rises significantly after age 60 years: approximately 8 per 100,000 between ages 60 and 70 years and greater than 20 per 100,000 after age 70 years.5 In 2004, close to 10,000 new US cases of MDS were registered, and approximately 7000 cases occurred in patients older than age 70 years.5 Prognosis is poor for patients with MDS, with 3-year survival rates estimated at less than 50%.5 The standard prognostic tool in MDS is the International Prognostic Scoring System (IPSS), which classifies patients into low-, intermediate-1, intermediate-2, and high-risk categories on the basis of the percentage of bone marrow blasts, the karyotype, and the number of cytopenias; the respective median survival rates are estimated at 8, 5.3, 2.2, and 0.9 years.6,7 Although additional IPSS-independent prognostic variables have since been described, their added practical value has been modest.8 From the standpoint of both disease biology and prognosis, it is important to distinguish primary MDS from therapy-related MDS; the latter is closely related to therapy-related AML and develops in the setting of prior exposure to chemotherapy (eg, alkylating agents, topoisomerase II inhibitors), radiotherapy, radiation accidents, benzene, or other toxins and is prognostically worse than primary MDS.9 Current management in MDS includes supportive care (eg, red blood cell transfusions), drug therapy, and allogeneic hematopoietic cell transplant (allo-SCT). Currently, allo-SCT is the only treatment modality for MDS with curative potential. However, because most patients with MDS are older than age 70 years,5 they are not good candidates for allo-SCT, and the value of allo-SCT in younger patients is undermined by the substantial risk of treatment-related death and morbidity.10-12 Furthermore, cure is not necessarily essential in the presence of drug therapy that can effectively control disease symptoms and prevent disease-related mortality.13,14 Unfortunately, drug therapy for MDS has had limited success, which reflects our inadequate insight into disease pathogenesis. Regardless, 4 new drugs (azacitidine, decitabine, lenalidomide, and deferasirox) were recently approved by the Food and Drug Administration for use in certain types of MDS. In the following sections, I comment on their true value in current practice.

Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

An 11-year-old female Dachshund was presented with depression, diarrhea, weight loss, and radiographic evidence of masses involving the liver, spleen, and cranial lobe of the right lung. Results of a CBC included severe nonregenerative anemia (HCT 14.2%, hemoglobin, 4.3 g/dL, reticulocytes 66,000/microL) with marked metarubricytosis (nucleated RBCs 6.39 x 10(3)/microL). Examination of the peripheral blood smear revealed marked erythroid dysplasia, including marked anisocytosis with a prevalence of macrocytes, Howell-Jolly bodies, diffuse basophilic stippling, and multinucleated and atypical nucleated RBCs. Neutrophil hypersegmentation and giant forms were also noted. Numerous erythrocytes, particularly polychromatophilic cells, contained inclusions consistent with Cabot rings, which appeared as delicate red-purple ellipsoid or figure 8 structures. Rarely, Cabot rings were observed extracellularly. The dog was treated symptomatically with blood transfusions, prednisone, erythropoietin, and vitamin supplementation, but the anemia progressively worsened. The dog was euthanized 2 months after presentation. Bone marrow aspirate and core biopsy specimens obtained at the time of euthanasia revealed marked dysplastic changes in all cell lines, especially dyserythropoiesis, along with infiltrating carcinoma cells. A necropsy was performed, and histologic examination revealed poorly differentiated adenocarcinoma of the lung with multiple metastases to the marrow, spleen, and liver. The final diagnosis was marked myelodysplasia secondary to metastatic adenocarcinoma. Cabot rings are found rarely in humans with myelodysplasia, but have not been described previously in dogs. Based on the findings in this case, Cabot rings may occur rarely in dogs with severe dyserythropoiesis.

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

Objective The aim of this study was to determine the expression of growth differentiation factor 15 (GDF-15) in patients with chronic hepatitis C virus and its association with iron-loading anemia in these patients. Background Significant shortened red cell survival has been reported in patients with liver diseases even in the absence of anemia. The mechanism for the observed decreased red cell lifespan is not fully understood, and it is most probably multifactorial. GDF-15, a member of the transforming growth factor-β super family, was first cloned from human monocytoid cell line and was found to inhibit tumor necrotizing factor production by macrophages and negatively regulate hepcidin. Recent studies suggest that GDF-15 levels increase in conditions of cell stress and apoptosis. In bone marrow (BM), GDF-15 increases in case of ineffective erythropoiesis. Patients and methods This study was carried out at Clinical Pathology Department, Faculty of Medicine and National Liver Institute, Menoufia University, in the period from May 2013 to May 2015. The study included 70 individuals: 50 patients with compensated chronic hepatitis C with unexplained anemia and 10 patients with idiopathic thrombocytopenic purpura who had normal red blood cell counts and no signs of erythroid dysplasia, as a control group for BM expression of GDF-15. In addition, 10 unrelated healthy age-matched and sex-matched adult individuals were included as a normal control group. Serum GDF-15 was measured by ELISA, and GDF-15 mRNA was measured by real-time RT-PCR. Results There were significant differences between the different studied groups regarding iron profile, GDF-15 serum level, and BM expression of GDF-15 mRNA, being higher in patients with chronic hepatitis C than other groups. GDF-15 was positively correlated with serum iron indices and negatively correlated with hemoglobin concentration. Conclusion Ineffective erythropoiesis contributes to iron overload and anemia in patients with chronic hepatitis C.

Erythroid dysplasia is a common feature of myelodysplastic syndromes (MDS). Currently available information about the immunophenotypic features of normal and dysplastic erythropoiesis is scarce and restricted to relatively few markers. Here we studied the expression of CD117, CD35 and CD44 throughout the normal (n=16) and dysplastic (n=48) bone marrow erythroid maturation. CD35 emerged as an early marker of CD34+ erythroid-committed precursors, which is expressed before CD105 and remains positive thereafter. MDS patients (with and without morphologic dyserythropoiesis) displayed overall increased expression of CD44, associated with slight alterations on CD35 expression, suggesting that phenotypic alterations in MDS may precede morphologic dysplasia. In turn, MDS patients with anemia showed increased expression of CD117., P.L. is supported by a grant from the Fundação para a Ciência e Tecnologia (FCT) of Portugal (SFRH/BD/32097/2006).

IMDSFlow19: et al., Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.