Megakaryocytic Dysplasia Research Articles

A systematic classification of megakaryocytic dysplasia and its impact on prognosis for patients with myelodysplastic syndromes.

BackgroundDys-megakaryopoiesis is defined as ≥10 % of dysplastic megakaryocytes in bone marrow smears by the World Health Organization. However, concordance rates for dysplastic megakaryocytes between different observers is low and, consequently, evaluation of dysmegakaryopoiesis is also often discordant.ResultsWe performed CD41 immune staining and proposed a systematic classification of dys-megakaryopoiesis on bone marrow films: (1) micro-megakaryocytes (<12 µm); (2) micro-megakaryocytes (12–40 µm) with 1 nucleus; (3) micro-megakaryocytes (12–40 µm) with 2 nuclei; (4) micro-megakaryocytes (12–40 um) with multiple (more than 2) nuclei; (5) dysplastic megakaryocytes (≥40 µm) with 1 nucleus; (6) dysplastic megakaryocytes (≥40 µm) with 2 nuclei; and (7) dysplastic megakaryocytes (≥40 µm) with multiple (more than 2) nuclei. Further, we evaluated the prognostic impact of micro-megakaryocytes and dysplastic mono-nucleated megakaryocytes on MDS patients. The best discriminator cut-off point for each group was determined by the minimal P value approach. In multivariate analyses micro-megakaryocytes ≥25 % and dysplastic mono-nucleated megakaryocytes ≥30 % were independent adverse prognostic factors (hazard ratio [HR] = 1.58 [95 % confidence interval [CI], 1.11, 2.23]; P = 0.010 and 1.53 [1.09, 2.16]; P = 0.014).ConclusionsOur data suggest integration of micro-megakaryocytes and dysplastic mono-nucleated megakaryocytes improve predictive accuracy of the international prognostic scoring system-revised (IPSS-R) scoring system.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-016-0041-6) contains supplementary material, which is available to authorized users.

Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-β1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-β1 signaling genes was altered in the marrow and spleen of PMF patients, respectively. Abnormalities included genes of TGF-β1 signaling, cell cycling and abnormal in chronic myeloid leukemia (EVI1 and p21(CIP)) (both marrow and spleen) and Hedgehog (marrow only) and p53 (spleen only) signaling. Pathway analyses of these alterations predict an increased osteoblast differentiation, ineffective hematopoiesis and fibrosis driven by non-canonical TGF-β1 signaling in marrow and increased proliferation and defective DNA repair in spleen. Since activation of non-canonical TGF-β1 signaling is associated with fibrosis in autoimmune diseases, the hypothesis that fibrosis in PMF results from an autoimmune process triggered by dead megakaryocytes was tested by determining that PMF patients expressed plasma levels of mitochondrial DNA and anti-mitochondrial antibodies greater than normal controls. These data identify autoimmunity as a possible cause of marrow fibrosis in PMF.

A Calcium-Dependent Pathway Determines Response to Lenalidomide in Del(5q) Myelodysplastic Syndromes

Deletions of the long arm of chromosome 5 (del(5q)) are common cytogenetic alterations in myelodysplastic syndrome (MDS), a disease characterized by refractory anemia, megakaryocyte dysplasia, and thrombocytosis. The thalidomide analogue lenalidomide (LEN) produces durable erythroid responses in ~60% of del(5q) MDS patients, including a majority of cytogenetic responses in which the del(5q) clone becomes undetectable in the bone marrow. Despite high response rates, clinical and cytogenetic relapse occur within 2-3 years. Mechanisms of clinical response, resistance and relapse with LEN therapy remain to be elucidated. The target of LEN has recently been identified as the cereblon (CRBN) component of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN). Upon LEN binding, the substrate-specificity of the CRL4-CRBN complex is altered, and LEN-regulated substrates are beginning to be identified.An RNA interference screen was performed to identify genes/pathways that mediate LEN sensitivity and resistance in del(5q) MDS. The LEN-sensitive del(5q) MDS patient-derived cell line MDSL was screened with a genome-wide shRNA library (SBI GeneNet Human 50K Library) in the presence and absence of LEN treatment (0 and 10 μM) for 7 days. Three independent shRNAs targeting the proton-sensing G protein-coupled receptor 1 (GPR68 or OGR1) were among the most enriched shRNAs in LEN-treated cells, suggesting that loss of GPR68 expression conferred resistance to LEN. This finding was validated in MDSL cells, using an independent set of shRNAs. Conversely, a GPR68 agonist (N-cyclopropoyl-5-[thiophen-2-yl]-isoxazole-3-carboxamid) enhanced LEN-induced cytotoxicity to MDSL cells. GPR68 is a proton-sensing G-protein coupled receptor that stimulates inositol phosphate production and/or intracellular calcium (Ca2+) mobilization. Curiously, CRBN was originally identified as a binding protein of calcium-activated potassium channels. These data led us to hypothesize that Ca2+ signaling may be responsible for LEN-mediated cytotoxic effect in MDS cells. Reducing intracellular Ca2+ level with chelators reversed LEN’s cytotoxic effects, while increasing intracellular Ca2+ level with ionomycin enhanced LEN’s cytotoxic effect, indicating that intracellular Ca2+ levels determine cellular responsiveness to LEN. Although LEN did not induce an instant burst of Ca2+ influx, a gradual increase of basal intracellular free Ca2+ was observed following LEN treatment in LEN-sensitive cell lines and primary MDS marrow cells, but not in LEN-resistant cells, suggesting that LEN cytotoxicity was dependent on the cell’s ability to release Ca2+ from intracellular stores. GPR68 and CRBN were both necessary for the LEN-induced increase in Ca2+, as knockdown of GPR68 or CRBN in LEN-sensitive cells prevented the Ca2+increase.To identify the Ca2+-dependent signaling pathway responsible for mediating the cytotoxic effect of LEN, a panel of seven inhibitors that blocked mitochondrial/caspase-, calpain-, autophagy-, or lysosomal-dependent cell death pathways was tested in combination with LEN on MDSL cells. Only the inhibitor of calpains (PD150606) prevented LEN-induced cytotoxic effects in MDSL cells, indicating that calpain activation was necessary for mediating cell death in LEN-treated cells. Calpains are Ca2+-dependent cysteine proteases that can induce apoptotic and necrotic cell death by proteolytic cleavage of protein substrates. Calpastatin, the only endogenous calpain inhibitor, is localized to 5q15 and its expression is haploinsufficient in del(5q) MDS as compared to normal karyotype MDS. Taken together, our results show that LEN increased intracellular Ca2+ levels by a CRBN- and GPR68-dependent mechanism, leading to calpain-mediated cytotoxicity in del(5q) MDS cells. We propose a model in which haploinsufficient expression of calpastatin in del(5q) MDS sensitizes cells to cytotoxic effects of LEN. Further studies are required to identify the direct LEN-modulated substrates of CRBN that mediate this effect. DisclosuresOliva:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria. MacBeth:Celgene: Employment, Equity Ownership. Starczynowski:Celgene: Research Funding.

Flow Cytometric Immunophenotyping in Myelodysplasia: Discovery and Diagnosis

Diagnosis of myelodysplastic syndromes (MDS) is based on the integration of results from diagnostic tools. Initial laboratory assessments in patients with suspected MDS comprise analysis of peripheral blood and bone marrow with the gold standard of cytomorphology, conventional cytogenetics and interphase fluorescence-in-situ hybridization (FISH). Single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) are emerging techniques. The pathological hallmark of MDS is dysplasia. Flow cytometry (FC) can identify aberrancies in antigen expression and differentiation patterns that are indicative of dysplasia. FC is regarded instrumental and now even recommended in the diagnostic work-up of (suspected) MDS, especially when dysplasia by cytomorphology is minimal and cytogenetics shows no (typical) abnormalities. No single FC marker has been identified that is specific for MDS. The WHO-2008 classification recommends the presence of three or more FC aberrancies as highly suggestive for MDS. Minimal requirements to analyze dysplasia by FC have been proposed by the European LeukemiaNet (ELNet) working group (ELNet iMDS-Flow. ELNet recommendations should enable a categorization of FC results in cytopenic patients as "normal", "suggestive of", or “high probability of” MDS. FC as a single technique is not sufficient for the diagnosis of MDS and should be part of an integrated diagnostic work-up. Within the ELNet-iMDS-Flow group, a score based on four cardinal parameters was validated in MDS patients with <5% blasts and non-clonal cytopenic controls. This diagnostic score comprises: a) SSC of granulocytes (reflecting granularity) as ratio to lymphocytes; b) percentage of CD34+ myeloid progenitors of nucleated cells; c) percentage of B cell progenitors within the total CD34+ compartment; and d) expression of CD45 on CD34+ myeloid progenitors as ratio to lymphocytes. An abnormal value for every single parameter is assigned 1 point; MDS is highly suggestive when a score of 2 or more is obtained. Sensitivity of this diagnostic score was 70% and specificity 92%. It was demonstrated that this score also separates distinct subgroups with respect to prognosis within IPSS-R risk groups. Most flow scores mainly incorporate markers that cover the myelomonocytic lineage, e.g. the flow cytometric scoring system (FCSS). The latter separates patients with no and mild-to-moderate dysplasia from those with severe dysplasia. The FCSS has recently been shown to add significantly in separating patients into low or high risk disease in the revised IPSS subgroups. Finally, FCSS originally designed as a prognostic score, but can also be applied as a diagnostic score as combined with the 4-parameter FC diagnostic score to further improve sensitivity and specificity. Flow profiles based on the myelomonocytic lineage may fail to recognize MDS patients that exclusively show erythroid and/or megakaryocytic dysplasia. Analysis of megakaryocytes is hampered by their paucity. Recent advances shows that when adding erythroid markers such as CD71, CD36 and CD117/CD105 may add significantly to the diagnostic score of MDS by FC. Interestingly, aberrancies in the myelomonocytic lineage have been shown in patients with solely erythroid dysplasia with impact on prognosis. In addition, aberrant FC profile of myeloid progenitors has been associated with high transfusion requirements and disease progression as well as with a short duration of response or even lack of response to growth factor and azacitidine treatment. In conclusion, FC is a useful tool in the diagnostic work-up of MDS. Further studies on the value of FC in diagnosis, prognosis and predicting response to treatment are ongoing, as well as correlation of FC results with data obtained by SNP and NGS within prospective multicenter clinical trials in low and high risk MDS. DisclosuresVan de Loosdrecht:celgene: Honoraria, Research Funding; alexion: Research Funding.

MiRNAs, Immune Signaling, and Myelodysplastic Syndromes Pathogenesis

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell hematologic malignancies characterized by cytopenias as a result of ineffective hematopoiesis and a propensity to progress to acute myeloid leukemia. In low-risk MDS, a characteristic finding in the bone marrow is that of increased apoptosis. The most common structural genomic aberration observed in MDS is the interstitial deletion of the long arm of chromosome 5. MDS with isolated del(5q) is a subtype of MDS characterized by severe anemia and variable neutropenia, but normal or high platelet counts with dysplastic megakaryocytes. MicroRNAs (miRNAs) are short noncoding RNAs capable of exerting their effects by postranscriptionally regulating numerous mRNA targets. We have shown that deletion of chromosome 5q correlates with loss miR-145 and miR-146a that are abundant in hematopoietic stem/progenitor cells (HSPC). Genes involved in innate immune signaling are significantly overrepresented when predicted targets of these two miRNas are surveyed. Specifically, Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) are targets of miR-145 and miR-16a, respectively. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6, to activate innate immune signaling in mouse HSPC, results in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow, in order to aberrantly activate innate immune signaling, progress either to marrow failure or acute myeloid leukemia. Loss of these miRNAs and consequent inappropriate immune signaling results in suppression of HSPC with a relatively greater effect on normal HSPC. Thus, inappropriate activation of innate immune signaling in HSPC phenocopies several general clinical features of low-risk MDS and of del(5q) MDS in particular. Recent work from our group defines additional cytokine pathways that are dysregulated by loss of miR-143 and miR-145. The impact of cytokine dysregulation on HSPC and the marrow microenvironment will be discussed. Disclosures Karsan: Celgene: Research Funding.

Somatic Mutations in Splicing Factor 3b, Subunit 1 (SF3B1) Are a Useful Biomarker to Differentiate Between Clonal and Non-Clonal Causes of Sideroblastic Anemia

Background: Sideroblastic anemia (SA) can present as congenital SA (CSA), acquired clonal SA, and acquired reversible SA. Patients (pts) with SA have anemia and ring sideroblasts (RS). Acquired clonal SA is often linked to myelodysplastic syndromes (MDS) or myelodysplastic/ myeloproliferative neoplasms (MDS/MPN). Clinico-pathologic overlap features, unmet morphologic and/or cytogenetic criteria complicate the diagnosis of SA leading to delayed therapies. Currently the diagnosis of SA is based on bone marrow (BM) examination and routine blood tests. There is a need to find easily testable biomarkers that can lead to faster diagnosis of clonal and non-clonal SA. Somatic mutation in splicing factor 3b, subunit 1 (SF3B1) are frequent in MDS-RS and MDS/MPN-RS and have been closely associated with RS.Objective:SF3B1 mutations can be a useful diagnostic biomarker for pts with acquired clonal SA who present with cytopenias and/or minimal morphologic changes suspicious of MDS and MDS/MPN but not sufficient to make a definitive diagnosis.Patients and Methods: Six pts with SA at presentation and seen at Cleveland Clinic were included in this study. The median age was 38 years (range, 6-75). Blood tests and BM biopsy showed persistent anemia [Hgb, 10.5 g/dL (range, 8.8-13)], RS [numerous (3 pts), 15% (1 pt), rare (1 pt) and >15% (2 pts)], 3/6 pts had minimal erythrodysplasia with 1 pt having a mild megakaryocytic dysplasia, 3 pts had hypercellular (60-90%), 2 pts had normocellular (50%, 80%) and 1 pt had hypocellular BM (30%) for age, and < 5% BM (2%=2 pts; 1%=1 pt; 3%=1 pt). Two pts had PLT count > 400 k/uL, 3 pts had > 100 k/uL, and 1pt <100 k/uL. Leukocytosis was observed in 2, leukopenia in 1, and neutropenia in 2 pts. Iron studies were unremarkable Molecular analysis was performed on SF3B1 (all exons), PRPF8 (exons 15, 25, 33), U2AF1 (exons 1, 6, 7), SRSF2 (exons 1, 2), DNMT3A (18-23), EZH2 (exons 18, 19) and IDH1/2 (exon 4). BCR/ABL p210 transcripts were assessed by RT-PCR. JAK2 (exon 14) was assessed by melting curve analysis.Results: All pts presented with sustained anemia, RS w/o or with minimal BM dysplasia, and normal karyotype. SF3B1 mutations (K666N and K700E) were detected in 2 pts (pts#2 and 6) before the clonal disease manifested. The first pt was a 75-year-old man with leukocytosis, transfusion-independent anemia, and fatigue. At the time of SF3B1 molecular testing the BM was hypercellular (60%) with <10%, megakaryocytic dysplasia, 2% blasts and 15% RS. JAK2 and BCR-ABL were wild type (WT). Later on, the patient developed hepatomegaly, his cytopenias worsened requiring RBC transfusions, and a repeat BM biopsy showed more prominent dysplasia of erythroid cells and megakaryocytes. The second pt was a 51-year-old man with fatigue, low WBC= 3.0 k/uL, ANC=0.7 k/uL, and PLTs=113 k/uL and macrocytic anemia (MCV=106 fL and Hgb=12.2 g/dL). His spleen was borderline enlarged (14 cm). BM biopsy showed 90% cellularity, erythroid hyperplasia, rare dyserythropoiesis and 60% RS. Suspicion for a possible MDS was raised although morphology was inconclusive. SF3B1 mutation (K700E) confirmed the clonality of the disease. A few months later, the pt developed worsening counts and overt MDS was confirmed by BM biopsy. All SF3B1WT pts were WT also for other genes known to be mutated in MDS and MDS/MPN like PRPF8, U2AF1, SRSF2, DNMT3A, IDH1/2, and EZH2. Only genetic tests showed an ALAS2 mutation in pt#1 and a paternally inherited duplication of 10q23.31 in pt#3. Both pts were diagnosed with CSA. Clinical history and work up assessed the same diagnosis for pt#4. Pt#5 presented with neutropenia and anemia. BM biopsy showed no dysplasia, 50% BM cellularity, trilineage maturation, and 25% RS. The presence of RS appeared to be induced by the use of an antibiotic prior the BM biopsy. Patient’s cytopenias and RS resolved after drug cessation.Conclusion: This case series although conducted in few pts provides important insights in the diagnostic use of molecular genetics in clinical practice. Often SA pts come to the clinic with inconclusive morphologic features of MDS or MDS/MPN. The presence of SF3B1 mutations may serve as an additional tool that can help differentiating between clonal and non-clonal cases of SA. In both pts, SF3B1 mutations anticipated the subsequent overt manifestation of clinical MDS or MDS/MPN phenotype. The collection of a larger cohort of pts is ongoing in order to further confirm this finding. DisclosuresNo relevant conflicts of interest to declare.

Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYKwt, TEL–SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYKwt enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK–SYK caused lethal T-cell lymphomas and the cytoplasmic TEL–SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL–SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL–SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL–SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

Minimal morphological criteria for defining bone marrow dysplasia: a basis for clinical implementation of WHO classification of myelodysplastic syndromes

The World Health Organization classification of myelodysplastic syndromes (MDS) is based on morphological evaluation of marrow dysplasia. We performed a systematic review of cytological and histological data from 1150 patients with peripheral blood cytopenia. We analyzed the frequency and discriminant power of single morphological abnormalities. A score to define minimal morphological criteria associated to the presence of marrow dysplasia was developed. This score showed high sensitivity/specificity (>90%), acceptable reproducibility and was independently validated. The severity of granulocytic and megakaryocytic dysplasia significantly affected survival. A close association was found between ring sideroblasts and SF3B1 mutations, and between severe granulocytic dysplasia and mutation of ASXL1, RUNX1, TP53 and SRSF2 genes. In myeloid neoplasms with fibrosis, multilineage dysplasia, hypolobulated/multinucleated megakaryocytes and increased CD34+ progenitors in the absence of JAK2, MPL and CALR gene mutations were significantly associated with a myelodysplastic phenotype. In myeloid disorders with marrow hypoplasia, granulocytic and/or megakaryocytic dysplasia, increased CD34+ progenitors and chromosomal abnormalities are consistent with a diagnosis of MDS. The proposed morphological score may be useful to evaluate the presence of dysplasia in cases without a clearly objective myelodysplastic phenotype. The integration of cytological and histological parameters improves the identification of MDS cases among myeloid disorders with fibrosis and hypocellularity.

Morphological changes in the bone marrow of the dogs with visceral leishmaniasis.

The aim of this study was to evaluate the most frequent lesions in the bone marrow of dogs naturally infected by Leishmania (Leishmania) chagasi. Thirty-three dogs sacrificed at the Zoonosis Control Center of Araçatuba, a municipality endemic for visceral leishmaniasis (VL), were used. The animals were classified as asymptomatic, oligosymptomatic, and symptomatic groups. At the necropsy, bone marrow samples were collected from the femur, fixed, processed, and stained with hematoxylin and eosin. The lesion intensity was classified as mild, moderate, or severe. The parasite load was determined using immunohistochemistry. The most important lesions consisted of multifocal to diffuse granulomas, megakaryocytic dysplasia, and medullary aplasia. There were no statistical differences between the three clinical groups regarding parasite load and lesion intensity. Asymptomatic dogs also presented high parasitism in the bone marrow as dogs with clinical signs of VL. It was concluded that, regardless of clinical group, the bone marrow is a site for multiplication of Leishmania chagasi. Possibly, the bone marrow dysplasia may arise from the presence of many parasitized and activated macrophages in this organ. Consequently, it affects the profile of hematopoietic cells in the bone marrow and systemic circulation.

Sustained Erythroid Response Associated With Red Cell Transfusion and Progressive Growth Factor Independence Is Feasible With Adjuvant Sertraline In Low-Risk Myelodysplastic Syndrome (LR-MDS)

BackgroundLR-MDS is a heterogeneous group of clonal marrow disorder associated with ineffective hematopoeisis and complex genomic etiology. For patients (pt) with refractory cytopenia phenotype (RC-P), transfusion and growth factor support, such as Erythropoietin Stimulating Agents (ESAs) and Granulocyte Colony Stimulating Factor (G-CSF), are treatment options. Response rates for ESA is about 30%, (and response potentiation can be achieved in about 35% of pt receiving concurrent G-CSF.However, ESA unresponsiveness is a common phenomenon and is associated with transfusion dependence, normal/high EPO levels, and high IPSS score. Therefore, novel therapies are greatly needed. Biologically, LR-MDS is linked to deregulation of apoptosis and cytokine signaling pathways. Selective Serotonin Reuptake Inhibitors (SSRIs) can induce normalization of deranged cytokines such as IL6 and TNF-α in clinical depression. In animal model of arthritis, sertraline abrogates manifestation of autoimmune arthritis by reducing TNF α. Our group recently showed that significant improvement in overall survival (OS) can be achieved in low-risk MDS patients treated with SSRIs, such as sertraline, which is independent of concurrent MDS directed therapy. (Li, et al. ASH 2012 abstract # 3818) Here, we report a case of sequential reversal of transfusion and growth factor dependence phenotype in a pt with refractory anemia multi-lineage dysplasia LR- MDS. MethodsA 72-year male with transfusion dependent refractory anemia (8 units RBC in 8 weeks) was evaluated in clinic. His bone marrow showed erythroid and megakaryocytic dysplasia consistent with RCMD by WHO 2008 classification. Cytogenetic study revealed 45;X-Y[4];46 XY[16]. R-IPSS=1.5 (Cytogenetic=0; blast [0%] =0; Hb=7g/dL=1.5; Platelets [135000] =0; ANC [1500] =0). EPO and ferritin levels were 449 IU/L and 1600 NG/ML, respectively. R-H-EPO was administered at 40000 IU subcutaneously (SQ) per week. In view of severe depression, pt initiated sertraline at 100 mg orally on day (D) 61 of treatment. Response after 12 weeks of ESA, sequential addition of G-CSF and sertraline, were assessed according to internal working group (IWG) 2006 criteria. ResultsAfter 3 months of standard weekly SQ ESA (D1-D92) (Fig.1), and given ESA refractoriness, G-CSF at 480 mcg SQ 5-times weekly was added (ESA + G-CSF schedule) (D92-D189).Transfusion independence was observed by D 161 of combined ESA+G-CSF(Fig.1). Progressive hemoglobin stabilization resulted in G-CSF dose reduction to twice a week from D189 –D 257 (Fig. 1). Sequential resolution of ESA dependence was observed from D257- D515. Over the treatment period, the absolute increase in Hb level was 4.2 g/dL from baseline. Restoration of ESA erythroid response was observed by D93-210 after G-CSF and sertraline addition. (Table. 1) Sustained and improved Hb levels were achieved after 200 days of G-CSF discontinuation and ESA dose de-escalation while on sertraline treatment. [Display omitted] ConclusionsReversal of transfusion dependence represents a critical step in treatment algorithm of LR-MDS pt presenting with refractory anemia. In this context, ferritin normalization has previously been recognized as independent prognostic factor for outcome in MDS. Our case suggests feasibility of restoration of ESA sensitivity with concurrent G-CSF+ sertraline, and evidence for sustained beneficial effect of sertraline while ESA de-escalation, which seems independent from G-CSF. The MDS disease modifying effect of sertraline in LR- MDS patient presenting with refractory anemia and experiencing ESA refractoriness should be evaluated in prospective trials. Disclosures:No relevant conflicts of interest to declare.

GATA2 Mutations In Pediatric Myelodysplastic Syndromes and Bone Marrow Failure

IntroductionInherited bone marrow failure syndromes (IBMFS), idiopathic aplastic anemia (AA), and myelodysplastic syndromes (MDS) represent a spectrum of bone marrow failure (BMF) conditions for which the underlying genetics and pathophysiology is still poorly understood. Heterozygous germline mutations in GATA2 have recently been described in three distinct conditions that include familial MDS/AML, Emberger syndrome and MonoMac syndrome, each of which exhibits great clinical heterogeneity.The Pediatric MDS and BMF Registry was established in 2010 to carefully characterize clinical and histopathologic phenotypes and investigate the molecular basis for these disorders. To date 158 eligible patients/probands and 28 family members have been enrolled. The goal of this study was to determine the prevalence of GATA2 mutations in pediatric patients with MDS and BMF and characterize their clinical and histopathologic phenotypes. Materials and MethodsSanger sequencing of GATA2 was initially performed on 3 families with a history of familial MDS and 103 patients with sporadic appearing primary MDS, AA or an unclassified BMF enrolled in the Pediatric MDS and BMF Registry. Family members were assessed in patients with pathogenic mutation to determine if the disease was inherited or sporadic. Mutations were confirmed in somatic and germline tissue wherever possible. IBMFS were ruled out by molecular testing. Rigorous phenotype analysis included clinical and laboratory data, and standardized centralized pathology review. Whole exome sequencing (WES) was performed on a subset of patients to evaluate additional cooperating mutations and possible secondary somatic events and clonal evolution. Possible candidate genes were verified by Sanger sequencing. ResultsWe identified pathogenic GATA2 mutations in a total of 16 individuals, including 12 patients (7 familial MDS cases and 5 sporadic MDS/BMF cases) and 4 first-degree relatives from 5 kindreds. Most mutations clustered in zinc finger 2. Previously identified mutations such as N371K and R396Q as well as novel point and frame shift mutations were identified. The median age at diagnosis was 15 years. There was strong male predominance (n=11). The clinico-pathologic diagnoses were RAEB/AML (n=4), refractory cytopenia of childhood (n=6) and MonoMac/other (n=6). Two out of the four families presented with features of Emberger syndrome. Two individuals presented with characteristic features of MonoMac Syndrome, of which one also showed bone marrow failure and pulmonary fibrosis suggestive of telomere disease. Very short telomeres (below the first percentile) were detected in all lymphocyte subsets consistent with dyskeratosis congenita (DC). However, genetic analysis did not reveal any of the known DC associated genes. Other associated pathology included severe gastrointestinal bleeding (n=2), severe polyneuropathy (n=2) and other cancers (n=1). A morphologically distinctive megakaryocytic dysplasia was a characteristic finding on histopathology. Monosomy 7 was the most common acquired cytogenetic abnormalities (n=6). Given this association we identified several additional individuals with MDS and monosomy 7 from our pathology archives and identified 2 additional patients with pathogenic GATA2 mutations. Secondary somatic mutations identified by WES included ASXL1. Thirteen out of the 14 pediatric patients with GATA2 mutations underwent hematopoietic stem cell transplant (HSCT). Ten out of these 13 patients are alive. ConclusionGATA2 mutations occur at a higher frequency than previously anticipated in pediatric MDS, and BMF, often occur sporadically and are associated with monosomy 7. While the clinical presentation is heterogeneous, the histopathologic features are often unique. Somatic genetic alterations likely play a role in clonal evolution. Given its significant implications for treatment decisions and donor selection GATA2 mutation screening should be performed on all patients with MDS, AA, and BMF disorders excluding classical IBMFS, and potential related donors. Disclosures:No relevant conflicts of interest to declare.

A case report of AML with myelodysplasia-related changes with aggressive course in association with t(3;8)(q26;q24)

Myelodysplastic syndrome (MDS) is frequently associated with clonal cytogenetic abnormalities which are major predictors of the disease outcome. t(3;8)(q26;q24) was reported as a rare recurrent cytogenetic abnormality in MDS/acute myeloid leukemia (AML) with poor prognosis. Here, we present a case of MDS which rapidly advanced to AML with t(3;8)(q26;q24) in an elderly patient. Anemia and prominent megakaryocytic dysplasia in the absence of marked thrombocytopenia were distinct morphologic features. Patient progressed to leukemia and succumbed to the disease within 7 months from the initial diagnosis. Morphologic and clinical features in this patient resembled “3q21q26 syndrome” which is also associated with poor prognosis. Reports of unfavorable outcome in the patients with t(3;8)(q26;q24) highlight the probable significance of this cytogenetic abnormality for risk stratification in MDS/AML.

Differential diagnosis of myelofibrosis based on WHO 2008 criteria: Acute panmyelosis with myelofibrosis, acute megakaryoblastic leukemia with myelofibrosis, primary myelofibrosis and myelodysplastic syndrome with myelofibrosis

The aim of this study was to characterize clinicopathological features of acute panmyelosis with myelofibrosis (APMF), acute megakaryoblastic leukemia with myelofibrosis (AMKL-MF), primary myelofibrosis (PMF) and myelodysplastic syndrome with myelofibrosis (MDS-MF) in order to provide the keys to the differential diagnosis of bone marrow (BM) fibrosis. We compared age, gender, splenomegaly, serum lactate dehydrogenase level, blood cell counts, blast counts in peripheral blood (PB) and BM, megakaryocyte counts, BM cellularity, dysplasia, and the karyotypes of patients with APMF (n=6), AMKL-MF (n=7), PMF (n=44), and MDS-MF (n=44). APMF showed hyperplasia of all three lineages, increase in megakaryocyte count with dysplasia and frequent abnormal karyotypes. AMKL-MF was associated with elevated BM blast counts, decreased BM megakaryocyte count with rare megakaryocytic dysplasia and chromosome 21 abnormality. PMF patients displayed splenomegaly, rare blasts in PB/BM, and JAK2 V617F mutation. MDS-MF patients showed pancytopenia, dysplasia in all three lineages and recurrent chromosomal abnormalities involving chromosome 5,7,12, and 17. Although differential diagnosis among APMF, AMKL-MF, PMF, and MDS-MF is very challenging due to the overlapping clinical and morphological features, meticulous investigation of the patient with respect to splenomegaly, blood cell count, PB and BM findings, and karyotype will serve as a guide to correct diagnosis.

A novel t(3;12)(q21;p13) translocation in a patient with accelerated chronic myeloid leukemia after imatinib and nilotinib therapy.

The acquisition of secondary chromosomal aberrations in chronic myeloid leukemia (CML) patients with Philadelphia chromosome-positive (Ph+) karyotype signifies clonal evolution associated with the progression of the disease to its accelerated or blastic phase. Therefore, these aberrations have clinical and biological significance. T(3;12)(q26;p13), which is a recurrent chromosomal aberration observed in myeloid malignancies, is typically associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and extremely poor prognosis. We have identified a recurrent reciprocal translocation between chromosomes 3 and 12 with different breakpoint at bands 3q21 in the malignant cells from a 28-year-old man. The patient was initially diagnosed as having Ph+ CML in the chronic phase. The t(3;12)(q21;p13) translocation occurred 4 years after the patient was first diagnosed with CML while undergoing tyrosine kinase inhibitor therapy. We confirmed the t(3;12)(q21;p13) translocation via fluorescence in situ hybridization assay by using whole-chromosome paint probes for chromosomes 3 and 12. Our findings demonstrate that, similar to other recurrent translocations involving 3q26 such as t(3;3) and t(3;21), the t(3;12)(q21;p13) translocation is implicated not only in myelodysplastic syndrome and acute myeloid leukemia but also in the progression of CML. These findings extend the disease spectrum of this cytogenetic aberration.

Iron chelation therapy for a case of transfusion-independent MDS-RARS with significant iron overload

A 60-year-old man was referred to our institution for evaluation of anemia. He had not received medical examination or treatment for years and had no family history of hematological disorders. He used a moderate amount of alcohol daily (75–100 g ethanol per day) for more than a decade. Laboratory examination at the first visit showed white blood cells, 2,900/mm (71 % neutrophils, 20 % lymphocytes, 6 % monocytes, 3 % eosinophils, 1 % basophils); hemoglobin concentration, 7.6 g/dl; platelet count, 301,000/mm, MCV, 106 fl; MCH, 36.2 pg; MCHC, 34.2 %; reticulocyte count, 3.4 %; AST, 60 IU/l; ALT, 84 IU/l; LDH, 225 IU/l; direct bilirubin, 0.15 mg/dl; indirect bilirubin, 2.31 mg/dl; C-reactive protein 0.31 mg/dl; serum iron, 232 lg/dl; UIBC, 16 lg/dl; ferritin, 3,278 ng/ ml; and haptoglobin, 16 mg/dl. Serum concentrations of vitamins B6 and B12 and of folic acid were normal, and HBs antigen, HBs antibody, and HCV antibody were negative. Bone marrow (BM) examination revealed hypercellular marrow (245,000/mm) dominated by erythroblasts with megaloblastic changes. Myeloblasts included 1 % nucleated cells, and there was no apparent myeloid or megakaryocytic dysplasia. On iron staining, most erythroblasts were ringed sideroblasts (Fig. 1a). Chromosomal analysis found normal male karyotype. A tentative diagnosis of refractory anemia with ringed sideroblasts subtype of myelodysplastic syndrome (MDSRARS) was made. Metenolone acetate (10 mg/day) was initiated, but hemoglobin levels did not improve (Fig. 1b). The possibility of X-linked sideroblastic anemia (XLSA), a congenital disorder that is occasionally diagnosed later in life [1], was considered, and pyridoxal phosphate hydrate (90 mg/day) was administered orally, but was ineffective (Fig. 1b). In addition to very high serum ferritin (3.278 ng/ ml) and elevated serum transferrin saturation (93.5 %), mild hepatic dysfunction (AST 60 IU/l, ALT 84 IU/l) and mild hepatosplenomegaly with markedly high density of the liver on CT was observed at presentation (Fig. 1c). Thus, this patient was considered to have iron overload (IO), despite the fact that he had never been transfused, was negative for HCV infection, and had no signs of chronic inflammation. Neither glucose intolerance nor decreased cardiac function was observed. After 1 year from the first visit, despite significant reduction of alcohol intake, serum ferritin levels remained high, and the abnormalities in liver function remained unchanged (Fig. 1b). Considering the possibility that sustained IO in the liver may lead to hepatic cirrhosis and/or hepatocellular carcinoma [2], we initiated administration of an oral iron chelating agent (deferasirox 1,000 mg/day). Serum ferritin decreased steadily, falling to less than 500 ng/ ml in 19 months (Fig. 1b). AST and ALT also normalized, and the density of the liver on CT decreased from 100 to 70 HU. No hematological improvement was observed, either in the peripheral blood (PB) or in the BM. Thus, the clinical picture of this patient consisted of an unusual combination of sideroblastic anemia and clinically H. Ohashi H. Nagai Division of Hematology, National Hospital Organization Nagoya Medical Center, Nagoya, Japan

Extreme Thrombocytosis Under Azacitidine in Patients with Myelodysplastic Syndrome

AbstractAbstract 4961 Background:Platelet increase under azacitidine in patients with myelodysplastic syndrome (MDS) has been acknowledged as an early predictive factor of response to treatment. However, extreme thrombocytosis under azacitidine has not been reported. Methods:We studied consecutive patients with MDS or MDS/myeloproliferative neoplasm (MDS/MPN) who had platelet counts near or over 1, 000 G/L under azacitidine. Results:Four patients, sex ratio 1:1, with median age of 65 years, had extreme thrombocytosis under azacitidine. Baseline characteristics were: WHO classification RAEB-2/CMML-1/CMML-2 in 2/1/1 patients, median platelet count 248 G/L (<400 G/L in all), normal karyotype/+8, −9/−7 in 2/1/1 patients, IPSS low/int-2/high in 1/2/1 patients. None had reticulinic fibrosis or ring sideroblasts>15% at baseline. A median number of 8 cycles of azacitidine was administered. Individual platelet counts reached 2, 960 G/L, 800 G/L, 1, 188 G/L and 2, 740 G/L. Thrombocytosis occured early after treatment onset or resumption (Figure 1). Histologic findings under treatment were: Increased cellularity (N=4), micromegakaryocytes and other signs of megakaryocytic dysplasia (N=4), reticulinic fibrosis grade I and II in 1 and 2 patients, respectively. JAK2 V617F mutation was detected in 1 patient (with maximum platelet count of 2, 900 G/L) and was undetectable in the remaining patients. None had a thrombotic or hemorrhagic event. Two patients had a concomitant increase of WBC count. Response to azacitidine was CR, PR and stable disease in 1/1/2 patients. Three patients received hydroxyurea (HU) in addition to azacitidine and one patient underwent hematopoietic stem cell transplantation (HCT). AML transformation occurred in 1 patient 25 months after azacitidine onset. Median overall survival after azacitidine onset was 25 months. Conclusion:Extreme thrombocytosis of the range of essential thrombocytosis, with megakaryocytic dysplasia and hyperplasia, was noted under azacitidine in 4 patients with MDS-MDS/MPN and normal baseline platelet count. Hypothetically, azacitidine may induce the expression of critical genes of megakaryopoiesis or platelet release in patients with rare mutations. Notably, JAK2 mutation was detected in only one patient. Alternatively, demethylation could unmask an underlying unclassified MDS/MPN similar to RARS-T. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Conditional Deletion of Asxl1 Results in Myelodysplasia

AbstractAbstract 308Loss-of-function somatic mutations in Addition of Sex Combs Like 1 (ASXL1) occur in a subset of patients with myeloid malignancies, most commonly in myelodysplastic syndrome (MDS). In addition, germline mutations in ASXL1 are observed in patients with Bohring-Opitz Syndrome, a developmental disorder characterized by neurologic and skeletal abnormalities. Previous work characterizing a constitutive gene-trap Asxl1 model was notable for significant perinatal lethality of Asxl1−/− mice with evidence of an overt hematopoietic phenotype in surviving mice. Given the lack of detailed data on the hematopoietic phenotype of Asxl1 loss in vivo, we created a murine model for conditional knockout of Asxl1 for tissue- and temporal-specific deletion with 4 different Cre recombinase alleles.We crossed our conditional Asxl1 allele to EIIa-cre to generate mice with germline loss of Asxl1; this revealed embryonic lethality of mice with homozygous germline Asxl1 loss. Mice with heterozygous germline deletion of Asxl1 were viable and fertile although a significant proportion had cranio-facial abnormalities. Timed-sacrifice of pregnant mothers from heterozygous EIIa-cre Asxl1+/− crosses revealed that homozygous Asxl1−/− pups survived to 18–20.5 days post-coitus, with all embryos characterized by craniofacial abnormalities including anopthalmia, microcephaly, cleft palates, and mandibular malformations similar to those seen with Bohring-Opitz syndrome.We then generated mice with conditional Asxl1 deletion in the hematopoietic compartment by crossing floxed mice with Vav-cre recombinase mice for hematopoietic deletion at birth, and with Mx1-cre mice for inducible deletion of Asxl1. Deletion of Asxl1 in both systems resulted in complete loss of Asxl1 as assessed by Western blot. In both models we noted progressive development of leukopenia and anemia in mice with homozygous loss of Asxl1 compared to age-matched controls (Figure). This was associated with extramedullary hematopoiesis and morphologic evidence of myeloid, erythroid, and megakaryocytic dysplasia, similar to that observed in human MDS (Figure). Flow cytometry revealed a progressive increase in immunophenotypically-defined multipotent progenitors (Lineage-negative, Sca1+, cKIT+, CD150-, CD48+ cells) in bone marrow and spleen of knockout (KO) mice. Characterization of HSC’s from 6-week old Vav-cre Asxl1−/− mice using serial competitive transplantation revealed a competitive disadvantage with transplantation of SLAM+ cells from 6-week old KO mice. A subset of Vav-cre Asxl1−/− mice developed a transplantable, monocytic-like leukemia beyond 6 months of age which was characterized by both proliferative and dysplastic features. Restriction of Asxl1 deletion to the megakaryocytic compartment using Pf4-cre revealed an age-specific decline in platelet production in Pf4-cre Asxl1−/− mice compared to age-matched controls, and a concomitant increase in bone marrow megakaryocytes in KO mice.Consistent with previous in vitro studies, hematopoietic-specific deletion of Asxl1 was associated with a marked decrease in H3K27me3 as assessed by histone Western blots of murine splenocytes and H3K27me3 ChIP-Seq of myeloid progenitors from 1 year-old Vav-cre Asxl1−/− mice compared with littermate controls. RNA-Seq from myeloid progenitors was integrated with H3K4me3/K27me3 ChIP-Seq to identify gene targets of Asxl1 loss associated with myelodysplasia.Given that MDS patients frequently present with concomitant ASXL1 and TET2 mutations, Vav-cre Asxl1fl/fl mice were also crossed with mice bearing floxed alleles of Tet2. Mice with combined hematopoietic-specific deletion of both Asxl1 and Tet2 developed bone marrow failure and hastened death compared with age-matched, single-gene deleted counterparts at 25 to 40 weeks of age.The findings here reveal that hematopoietic-specific deletion of Asxl1 results in progressive ineffective hematopoiesis, an increase in hematopoietic progenitors, a propensity for leukemic transformation with age, and morphologic features of human MDS (Figure). Combining Asxl1 deletion with loss of Tet2, a combined genotype present in at least 5% of patients with de novo MDS, resulted in shorter latency, progressive MDS. These data suggest that these two models represent novel, genetically accurate models of MDS amenable to epigenomic, functional and preclinical therapeutic studies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Comparison of the Transforming Effect of Different SYK Oncogenes in Vivo Reveals TEL-SYK As Inducer of an MDS/MPS Overlap Syndrome in Mice

AbstractAbstract 1310The spleen tyrosin kinase (SYK) was shown to be overexpressed in various human hematologic malignancies like B-cell lymphomas and acute myeloid leukemias. There are two known translocations involving the SYK kinase: ITK-SYK and TEL-SYK. ITK-SYK was identified in unspecified peripheral T-cell lymphomas, whereas the TEL-SYK fusion had been discovered in a patient with myelodysplastic syndrome (MDS). Methods:All SYK containing oncogenes, ITK-SYK, TEL-SYK and SYK wt, were cloned into a pMSCV-IRES-GFP retroviral vector. All oncogenes and a GFP control vector were overexpressed in bone marrow of 5-flurouracil-pretreated mice and oncogene containing bone marrow was transplanted into irradiated recipient mice. Disease development was monitored over 1 year. Additionally the three SYK oncogenes were overexpressed in a myeloid (32D), B-lymphoid (BaF3) and T-cell lineage (Jurkat) to monitor cell context specific differences in cell signaling. Results:Here, we demonstrate that the overexpression of 3 SYK containing oncogenes, SYK wt, TEL-SYK and ITK-SYK in a murine bone marrow transplantation model induces the development of 3 different hematologic (pre)diseases. While ITK-SYK exclusively induced T-cell lymphomas in mice, we found that the TEL-SYK fusion induced an MDS/MPS overlapsyndrom. The mice developed an anemia and thrombopenia with signs of dysplasia of megakaryocytes and the erythroid lineage in the bone marrow and spleens. In addition those mice showed a strong expansion of the myeloid lineage, with leucocytosis in the peripheral blood mainly consisting of differentiated neutrophils. The bone marrow shows expansion of differentiated myeloid cells, but also myeloid progenitors and expansion of HSCs. Interestingly, also SYK wt induced a relative expansion of the myeloid compartment without inducing a leucocytosis or a lethal disease indicating a premalignant myeloproliferative state. Only TEL-SYK was able to induce IL-3 independent growth of BaF3- and 32D cells and could activate STAT5, STAT6, ERK, PLCγ2 and SLP76 in those cell lines. SYK and ITK-SYK were able to activate PLCγ2 and SLP76, but no STAT transcription factors. STAT5 activation by TEL-SYK was detectable also in vivo, which might indicate STAT5 as the major driver of TEL-SYK induced transformation.Our results demonstrate, that the various SYK oncogenes can induce different hematologic diseases in mice and reveales TEL-SYK as the first oncogene inducing an MDS/MPS overlap syndrome in mice. Disclosures:No relevant conflicts of interest to declare.

Enforced Expression of the Toll/Interleukin-1 Receptor Associated Protein in Murine Marrow Results in MDS-Like Bone Marrow Failure Mediated by Interferon Gamma

AbstractAbstract 555The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineage of the bone marrow, resulting in peripheral cytopenias and the propensity to develop into either acute myeloid leukemia (AML) or bone marrow failure (BMF). The molecular and cellular events involved in MDS development are yet to be elucidated. Large scale cytogenetic aberrations such as del(5q) have been associated with many types of MDS. Previous work from our lab has shown that the chromosomal band 5q32–33 harbours microRNAs (miR-145 and miR-146a), haploinsufficiency of which recapitulates the megakaryocytic dysplasia and neutropenia associated with del(5q) MDS. Ingenuity pathway analysis predicts that innate immune signaling is one of the most highly deregulated pathways by loss of these two miRNAs. Two innate immune signaling molecules, TNF receptor associated factor 6 (TRAF6) and Toll/interleukin-1 receptor associated protein (TIRAP), are targets ofmiR-146a and miR-145 respectively. While the role of TRAF6 in del(5q) MDS has been investigated, little is known about the role of TIRAP in MDS pathogenesis.To investigate the role of TIRAP in aberrant hematopoiesis, we transplanted lethally irradiated mice with bone marrow cells expressing TIRAP or vector control. TIRAP transplanted mice develop an MDS-like bone marrow failure characterized by anemia, neutropenia, and thrombocytopenia as early as 4 weeks post-transplant (unlike TRAF6 transplanted mice which succumb to BMF or AML approximately 4 months post-transplant). The discrepancy between TIRAP and TRAF6 disease progression models suggests that TRAF6 independent signaling originating from TIRAP may be responsible for the rapid bone marrow failure onset. Furthermore, TIRAP transplanted mice display hypocellular marrows characterized by increased apoptosis as measured by Annexin V/PI staining, similar to low risk MDS patients.In MDS, normal hematopoiesis is blocked in the non-disease fraction of bone marrow by autoimmunity associated cytokines. To identify factors that may be responsible for suppression of normal hematopoiesis in our BMF model, we performed cytokine expression profiling of TIRAP expressing marrow. Quantitative RT-PCR showed increased expression of both IL-10 and IFNγ in TIRAP expressing marrow compared to vector control. Interestingly, IFNγ is known to suppress hematopoiesis has been shown to be overexpressed in BM mononuclear cells in MDS and other BMF conditions. Also, IL-10 levels have been shown to be elevated in high risk MDS patients. To investigate the role of IL-10 and IFNγ in TIRAP mediated marrow failure, we transduced IL-10 −/− and IFNγ −/− marrow with TIRAP or vector control, and transplanted wildtype leathally irradiated mice. Loss of IFNγ but not IL-10 partially rescues the pancytopenia phenotype and prevents early death due to bone marrow failure. Interestingly however, mice transplanted with TIRAP expressing IFNγ −/− bone marrow succumb to a myeloproliferative disorder at later time points, suggesting that the factors responsible for myelosuppression are the same ones responsible for progression to AML. Disclosures:No relevant conflicts of interest to declare.

DNA methylation analysis of ALOX12 and GSTM1 in acute myeloid leukaemia identifies prognostically significant groups

To determine the role of DNA methylation in the progression of acute myeloid leukaemia (AML), we analysed the methylation status of ALOX12, GSTM1, HS3ST2 and FZD9 in 127 AML patients. Aberrant methylation of ALOX12 was associated with the subcategory AML with myelodysplasia-related changes (P=0·0439) and specifically with megakaryocytic dysplasia (P=0·0003). An association between HS3ST2 and AML patients with favourable cytogenetic risk was identified (P=0·0469). In univariate and multivariate analysis, methylation of GSTM1 was associated with worse overall survival (OS) and disease-free survival (DFS), with hazard ratios of 2·57 and 1·86, respectively. Furthermore, the significance of methylation of GSTM1 in predicting poor prognosis was maintained within the subcategories of AML not otherwise specified (NOS), AML with intermediate cytogenetic risk and normal karyotype AML. Finally, patients with both GSTM1 and ALOX12 methylated, demonstrated worse outcomes when all AML patients were assessed (OS; P=0·000411) as well as within AML NOS (DFS; P=0·0023), AML with intermediate cytogenetic risk (OS; P=0·0104) and normal karyotype AML (OS; P=0·00636). This study implicates methylation of specific genes in the classification and prognostication of AML and suggests that the morphological feature of multilineage dysplasia may be a surrogate marker of gene methylation in at least a subset of AML cases.