Early Myelodysplastic Syndromes Research Articles

Impact of Early Cytomegalovirus Reactivation After Allogeneic Hematopoietic Stem Cell Transplantation on Relapse in Patients With Myelodysplastic Syndrome: A Nationwide Retrospective Study From Adult Myelodysplastic Syndrome Working Group of the JSTCT

Cytomegalovirus (CMV) reactivation is a prominent complication associated with adverse outcomes in allogeneic hematopoietic stem cell transplantation (HSCT). However, CMV reactivation after allogeneic HSCT may be associated with a lower incidence of relapse in some hematological malignancies. This study analyzed the Japanese registry data from 1082 patients with myelodysplastic syndrome (MDS) who underwent their first allogeneic HSCT and survived for 100 days after transplantation without graft failure or disease relapse to investigate this association. Patients who received cord blood transplants, demonstrated in vivo T cell depletion, underwent prophylactic anti-CMV treatment, or diagnosed with secondary MDS were excluded. CMV reactivation measured by pp65 antigenemia within 100 days after allogeneic HSCT was observed in 57.5% of patients, with a median time of 46 days from transplant. The 5-yr overall survival and cumulative incidence of relapse (CIR) in the cohort were 60.5% and 15.6%, respectively. The 5-yr CIR showed no significant difference between patients with and without CMV reactivation (14.4% versus 17.2%; P = .185). Interestingly, CMV reactivation within 100 days was significantly associated with a lower 5-yr CIR (7.6% versus 16.4%; P = .002) in patients with <5% myeloblasts in the bone marrow (BM) just before HSCT. Furthermore, this relevancy confirmed even when excluding patients with Grade II to IV acute GVHD (Hazard ratio: 0.38; 95% confidential intervals: 0.18–0.801; P = .011). Our findings indicate a correlation between early CMV reactivation and MDS relapse, based on the proportion of myeloblasts in the BM. These results may contribute to the development of effective CMV prophylaxis post-HSCT.

Abstract 74: Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis

Abstract Increased cellular apoptosis is commonly seen in myelodysplastic syndrome (MDS), but how clearance of apoptotic cells, especially neutrophils, impacts disease progression is unknown. Moreover, while dysfunction of bone marrow (BM) stromal cell populations has been described in patients with MDS, the mechanisms inducing stromal defects are not well understood. Our laboratory found that BM macrophages are defective in a murine model of MDS (Vav-Nup98 HoxD13, aka NHD13), with decreased efferocytotic rates and increased inflammatory mediators. The increased apoptotic burden and defective macrophages would be expected to recruit non-professional phagocytes (osteoblasts) in the bone and bone marrow. Based on these data, we hypothesize that OBs (osteoblasts) participate in the clearance of apoptotic cells in the bone marrow microenvironment (BMME) to maintain BM homeostasis. However, the increased osteoblastic efferocytosis burden in MDS contributes to OBs’ dysfunction as a mechanism of MDS-dependent BMME disruption that may lead to MDS progression. We isolated from wild-type (B6) mice bone-associated cells (BAC), which are enriched with OBs. In vitro co-culture with labeled human end-stage neutrophils at 1:1 and 1:10 (OB: PMN) increased OB apoptosis, while senescence was increased only in high-dose PMN co-culture. We found that these changes were cell-autonomous. In vivo injection of neutrophils into WT mice showed around 5-10% efferocytosis rate in OBs. In efferocytotic OBs, apoptosis rates were significantly increased. Together, these data show that OBs participate in efferocytosis and that efferocytosis disrupts OBs, causing apoptosis and senescence. To mimic the MDS microenvironment, we used a genetic model where one of the Isocitrate dehydrogenase 2 (IDH2) mutations found in MDS (R140Q) is targeted to hematopoietic cells via the Vav promoter (IDH2R140Q mice) as well as NHD13 mice, representing early and late MDS BMME respectively. We observed that 6-to-8 months old NHD13 mice experienced more end-stage neutrophils[CL1] in the BMME (4.6% rate of end-stage neutrophils in WT compared to 9.5% rate in NHD13 mice), where OB apoptosis was also increased in vivo. In IDH2R140Q mice at 3 months of age, we confirmed the presence of mutated IDH2 in bone marrow cells by increased intracellular levels of the oncometabolite 2-hydroxyglutarate. At this early time point, IDH2R140Q mice did not have increased rates of apoptosis in CD45+ cells, and we found no increase in OB apoptosis. Analysis of IDH2R140Q mice at later time points is ongoing. Our data identify novel mechanisms to explain the relationship between defective myeloid cells and OB apoptosis in MDS that may contribute to the inflammatory MDS microenvironment, impact disease progression, and serve as future therapeutic approaches for MDS patients. Citation Format: Chunmo Chen, Emily R. Quarato, Yuko Kawano, Noah A. Salama, Hiroki Kawano, Michael W. Becker, Jeevisha Bajaj, Laura Calvi. Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 74.

SF3B1 mutations in myelodysplastic syndromes: A potential therapeutic target for modulating the entire disease process.

Myelodysplastic syndromes (MDS) are clonal hematologic malignancies characterized by ineffective hematopoiesis and dysplasia of the myeloid cell lineage and are characterized by peripheral blood cytopenia and an increased risk of transformation to acute myeloid leukemia (AML). Approximately half of the patients with MDS have somatic mutations in the spliceosome gene. Splicing Factor 3B Subunit 1A (SF3B1), the most frequently occurring splicing factor mutation in MDS is significantly associated with the MDS-RS subtype. SF3B1 mutations are intimately involved in the MDS regulation of various pathophysiological processes, including impaired erythropoiesis, dysregulated iron metabolism homeostasis, hyperinflammatory features, and R-loop accumulation. In the fifth edition of the World Health Organization (WHO) classification criteria for MDS, MDS with SF3B1 mutations has been classified as an independent subtype, which plays a crucial role in identifying the disease phenotype, promoting tumor development, determining clinical features, and influencing tumor prognosis. Given that SF3B1 has demonstrated therapeutic vulnerability both in early MDS drivers and downstream events, therapy based on spliceosome-associated mutations is considered a novel strategy worth exploring in the future.

Clinical and Biological Effects of Canakinumab in Lower-Risk Myelodysplastic Syndromes (MDS): Results from a Phase 2 Clinical Trial

Interleukin 1 beta (IL-1B) induced inflammasome activation is a mechanism of cancer initiation and progression (Schroder K, Cell, 2010). Our previous studies (Ganan-Gomez I, Blood 2021) showed that IL-1B was one of the most overexpressed pro-inflammatory cytokines in the hematopoietic stem and progenitor cells (HSPCs) of patients with clonal cytopenia of undetermined significance (CCUS) carrying DNMT3a and TET2 mutations. Given that IL-1B upregulation induces aberrant myeloid differentiation of HSPCs (Pietras EM, Nat Cell Biol., 2016), we hypothesize that IL-1B plays a critical role in MDS pathogenesis, and that targeting IL-1B signaling overcomes aberrant MDS HSPCs' myeloid skewing. To test this hypothesis, we designed a clinical trial to evaluate the IL-1B inhibitor canakinumab's (NCT04239157) safety profile and its clinical and biological effects in patients with lower-risk MDS. Patients and Methods: Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS, normal renal and hepatic functions, and performance status, who had previously received at least 1 line of other therapy were included in the study (Table1). Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. Bone marrow (BM) samples were obtained before therapy and after 1, 2, and 4 cycles of therapy during the trial and after every 3 or 4 cycles of therapy thereafter. Single-cell RNA sequencing (scRNA-seq; 10X Genomics) analyses were performed in lineage negative (Lin-)CD34+ HSPCs and mononuclear cells (MNCs) isolated at different therapy timepoints from the BM of 1 patient who achieved hematological improvement after canakinumab treatment, 2 patients who achieved stable disease, and 2 patients who did not respond to this therapy. The clinical characteristics of the 5 patients whose samples were analyzed by scRNA-seq analyses are included in Table 1. Results: Twenty-six patients have been enrolled in the clinical trial. Patients' clinical characteristics are included in Table 1. Twenty-three patients were evaluable for response. Among the 22 out of the 23 patients who were transfusion dependent at baseline, one achieved transfusion independence, and another had platelet count improvement. Three out of the 23 had progressive disease, and 18 had stable disease. Treatment was well tolerated with no severe adverse events. One patient died of sepsis unrelated to therapy. Overall, we did not observe any improvement in the blast counts and cytogenetic and/or mutational burden. To evaluate the biological effects of canakinumab, we performed scRNA-seq of Lin-CD34+ HSPCs (Figure 1) and MNCs at therapy baseline and after 2, 4, 5, 8, and 11 cycles of treatment. Consistent with our initial hypotheses, these analyses revealed that canakinumab decreased the frequencies of hematopoietic stem cells (HSCs) and multipotent myeloid progenitors (MPPs). Differential analysis between the samples showed that HSCs and MPPs significantly downregulated the expression of genes involved in cytokine signaling (e.g., IL-1B, CXCL2, and CXCL8), TNFα-induced activation of NF-κB, and hypoxia pathways, which demonstrated efficient target engagement of canakinumab on MDS cells. Similar results were also observed in downstream monocytes and B cells. In contrast, we did not observe any significant change in the HSPC architecture in the BM of patients with stable disease or whose disease did not respond to canakinumab. Consistent with our previous results in early stage MDS, the patient who achieved hematological improvement after canakinumab therapy was the only one among the cohort who carried a single mutation affecting DNMT3A (Table1). Conclusion. This study demonstrates that canakinumab is safe in MDS and efficiently targets IL-1B signaling. However, the biological effect of canakinumab has not yet translated in clinical responses in the subset of patients enrolled in this clinical trial. The study will accrue transfusion independent patients and patients with early stage MDS as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predictors of Disease Progression and Survival in Patients with Myelodysplastic Syndrome Secondary to Inherited Bone Marrow Failure Syndromes

Introduction Inherited bone marrow failure syndromes (IBMFSs) are rare genetic disorders characterized by abnormal hematopoiesis resulting in cytopenias, risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Once patients develop MDS, the only cure is hematopoietic stem cell transplant (HSCT). Proceeding to HSCT before transformation to advanced MDS or AML leads to better outcomes (Smith et al., PBC 2013). The rate of disease progression from early MDS to advanced MDS or AML is variable and risk factors for progression in IBMFS patients are unknown. We hypothesized that certain variables could predict the likelihood of progression, and that the type of disease progression may impact overall survival (OS). Methods Data were collected from patients prospectively enrolled in the Canadian Inherited Marrow Failure Registry (CIMFR), a Canadian collaboration including >95% of pediatric IBMFS patients. Patients were diagnosed with a specific syndrome or unclassified IBMFS (ucIBMFS) based on published criteria. Diagnostic criteria for pediatric MDS defined by Cada et al. (Haematologica 2015) were used, patients with refractory cytopenia (RC) with or without dysplasia or ringed sideroblasts were included. Patients with refractory cytopenia with excess blasts (RCEB) were excluded. MDS progression was defined as 1 or more of: a new cytogenetic abnormality, more advanced cytopathology, worse cytopenias requiring HSCT. Variables evaluated included: involvement of specific organ systems, hematologic parameters within 3 months of cytogenetic clones/MDS diagnosis, type of mutation (as per Waespe et al., Genomic Medicine 2017). Univariate Cox Proportional Hazard modelling was used to estimate association with specific variables and disease progression, Kaplan-Meier analysis was used for survival evaluation. Results Between January 1, 2001 and November 30, 2018, 601 patients were enrolled in the CIMFR, and during this time 59 (9.8%) developed cytogenetic clones/MDS. Four did not meet the pre-defined criteria for IBMFS, 9 had RCEB at diagnosis with MDS and 1 did not have data available; these were excluded from our cohort of 45 patients. Twelve (26%) patients with clones/MDS had Fanconi Anemia (FA), 13 (28.3%) had Shwachman-Diamond Syndrome (SDS), 5 (10.8%) had ucIBMFS, 15 (33.3%) had other IBMFSs. The most common cytogenetic finding at diagnosis was -7/-7q (n=10, 22%). Patients who developed clones/MDS had significantly worse OS compared to those without clones/MDS (HR 3, 95% CI 2-6, p<0.0001). Among patients who developed clones/MDS, 22 (48.9%) underwent HSCT. Twenty one clones/MDS patients (46.7%) had disease progression, with a median time to progression of 7.9 months (1.2-131.4). The majority (68.4%) developed progression within one year of clones/MDS diagnosis. Nine (42.8%) developed more advanced cytopathology, 9 (42.8%) a new cytogenetic abnormality and 5 (23.8%) worsening cytopenias. Most patients with FA (67%) had disease progression, compared to 38.5% of patients with SDS. Mutation types (classified as more vs less deleterious) were similar between the groups with and without progression. The evaluated hematologic parameters (MCV, hemoglobin, platelets and neutrophils within 3 months of diagnosis) were not predictive of disease progression. Patients with disease progression, when compared to those without, were more likely to have congenital malformations of the skin (14.3% vs 0%), cardiovascular system (28.6% vs 4.2%) and kidneys (14.3% vs 4.2%). No specific system involvement predicted disease progression, although congenital malformations of the cardiovascular system had a HR of 2.63 (95% CI 0.93-7.42). Conclusion Development of MDS has a significant adverse impact on the OS of IBMFS patients, with disease progression typically occurring within the first year after MDS diagnosis. Patients with FA have a high risk of disease progression. Hematologic parameters were not predictive of the risk of disease progression, however, the presence of congenital cardiovascular anomalies is suggestive of increased risk of disease progression and warrants further investigation. Our study is limited by the rare nature of IBMFSs and MDS and the collection of additional data will help to guide treatment decisions for this high-risk patient population. Acknowledgment The authors thank Mr. Bryan Maguire for his statistical analysis of data related to this work.

Microenvironment Profiling in Myelodysplastic Syndromes and Its Relationship to Clonal Hematopoiesis and Disease Progression

Microenvironment Profiling in Myelodysplastic Syndromes and Its Relationship to Clonal Hematopoiesis and Disease Progression

Pathophysiology of Myelodysplastic Syndromes

Ineffective hematopoiesis is the major characteristic of early myelodysplastic syndromes. Its pathophysiology relies on a diversity of mechanisms supported by genetic events that develop in aging hematopoietic stem cells. Deletion and mutations trigger epigenetic modifications, and co-transcriptional and post-transcriptional deregulations of gene expression. Epistatic interactions between mutants may aggravate the phenotype. Amplification of minor subclones containing mutations that promote their growth and suppress the others drives the clonal evolution. Aging also participates in reprogramming the immune microenvironment towards an inflammatory state, which precedes the expansion of immunosuppressive cells such as Tregs and myeloid-derived suppressive cells that alters the anti-tumor response of effector cells. Integrating biomarkers of transcription/translation deregulation and immune contexture will help the design of personalized treatments.

Programmed Necrosis Correlates with Development and Progression of Myelodysplastic Syndromes

Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow. Lower-grade MDS (bone marrow blasts <5%) is challenging to diagnose, as the morphologic dysplasia may be subtle and many patients lack characteristic cytogenetic abnormalities.Multiple studies of the bone marrow in MDS have demonstrated excessive hematopoietic cell death, and this increased cell death is thought to play an important role in the pathogenesis of the disease. Early studies of MDS revealed increased in situ end labeling and increased TUNEL staining which led to the conclusion of apoptotic cell death, but these methods cannot properly distinguish between necroptosis and apoptosis. Apoptosis is driven by caspases that break down the cytoskeleton to cause collapse of the cell in a predominantly immune silent process. Recently, necroptosis has been identified as a new pathway of programmed cell death, which is driven by Rip kinases and is characterized by premature rupture of the plasma membrane, resulting in an immune response. Necroptosis has been shown to play a role in inflammatory diseases as well as host response to infection, and tumors.Methods: To assess cell death status in normal bone marrow as compared to lower-grade MDS (RCUD, RA, RARS, RCMD) and higher-grade MDS (RAEBI/RAEBII, RAEB-t), we evaluated a total of 37 de novo MDS cases and 36 normal control cases. The MDS specimens included 20 cases of lower-grade MDS and 17 cases of higher-grade MDS. All cases were reviewed and the diagnosis confirmed by board-certified hematopathologists. Necroptosis was assessed by measuring Rip1 and phosphorylation of Mixed Lineage Kinase domain-like protein (MLKL) and apoptosis was measured by cleaved-caspase 3.Formalin fixed paraffin embedded bone marrow core biopsies were immunostained with the apoptosis marker (caspase 3) and necroptosis markers (Rip1, MLKL) and visualized by immunofluorescence. Whole slide images were obtained using Aperio Versa 200 (Leica Microsystems). We developed a pipeline for statistical analysis to evaluate the immunofluorescence of stained and scanned images using CellProfiler (Broad Institute) image analysis software. For each slide over 10,000 cells were analyzed.Results: We found that the proportion of RIP1 positive cells was significantly different between normal controls (6.91±1.32%) and lower-grade MDS (32.80±6.19%, p value 0.0042). Likewise, RIP1 staining in higher-grade MDS was less common than in lower-grade MDS (15.99±4.76% vs 32.90±6.19%, p value 0.0506). There was no significant difference between normal controls and high-grade MDS. In lower-grade MDS samples, RIP1 positive cells were mononuclear cells arranged in clusters, morphologically consistent with erythroid progenitors. Similar trends were seen with MLKL staining.To evaluate apoptosis, we performed cleaved caspase 3 staining on the same patient samples. There was no difference in the proportion of caspase 3 positive cells among the groups: normal controls (2.02±1.08%), lower-grade MDS (2.77±0.86%) or higher-grade MDS (1.99±0.71%). As apoptotic and necroptotic cells have markedly different cellular ultrastructural morphologies, we performed electron microscopy on fresh bone marrow cells to define cell death. After scoring cells with a nucleus as either being live, apoptotic (shrinkage of cell membrane and pyknotic nucleus), or necrotic (swelling of cell membrane and nucleus), we found that there was a significant increase in the number of necrotic cells in the bone marrow of the lower-grade MDS patients relative to the normal control or higher-grade MDS patients.We then performed over-representation analysis of the publicly available MDS microarray data (GEO GSE15061) from the international multi-institutional Microarray Innovations in Leukemia (MILE) research program. Although the study was underpowered to identify differences between normal controls and early MDS, we found that RIP1, RIP3 and MLKL had significantly increased expression in the MDS group vs the de novo AML group, suggesting that genes in the necroptotic pathway may be upregulated in MDS as compared to AML.In summary, our findings reveal that the necroptosis cell death pathway is upregulated in early MDS relative to normal controls and warrants further study to define its role in the pathogenesis of MDS and as a potential biomarker for the diagnosis of lower-grade MDS. [Display omitted] DisclosuresSavona:Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Incyte Corporation: Consultancy, Research Funding; Karyopharm: Consultancy, Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Modeling ASXL1 Mutation Revealed Myelodysplasia Caused By Derepression of p16Ink4a through Aberrant PRC1-Mediated Histone Modification

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant condition in patients with blood cells that possess somatic mutations of genes known to be recurrently mutated in hematologic malignancies. Among them is additional sex combs like 1 (ASXL1), whose mutation is frequent in various myeloid malignancies and associated with poor prognosis, and ASXL1 is known to act as an epigenetic modulator. Despite the clinical impact of ASXL1 mutations in myeloid malignancies, lack of animal models expressing mutated Asxl1 at endogenous levels mimicking human ASXL1-mutated diseases hinders from dissecting precise molecular mechanisms of mutated Asxl1. Here in this study, with newly generated knock-in mice carrying a frameshift mutation at the exon 13 of the Asxl1 gene (Asxl1G643WfsX12), which is identical to the most frequent human ASXL1 G646WfsX12 mutation, we extensively investigated the biological effects of the mutant.Asxl1G643fs/+ mice showed decreased body size and weight, resembling hypomorphic features in patients with Bohring-Opitz syndrome carrying de novo constitutive ASXL1 mutation.Asxl1G643fs/+ mice developed slowly progressive peripheral leukopenia, and Wright-Giemsa stain of bone marrow (BM) cells from Asxl1G643fs/+ mice showed dysplastic myeloid cells represented by hypersegmented neutrophils, apoptotic neutrophils and hyposegmented neutrophils with fine nuclear bridging consistent with pseudo-Pelger-Huët anomaly. Flow cytometric analyses of Asxl1G643fs/+ BM cells confirmed aberrant myeloid differentiation with significant increase of granulocytic and monocytic cell fraction, significant decrease of hematopoietic stem cell (HSC) pool (c-kit+ Sca-1+ Lin- (LSK) cells and CD34- CD135- LSK cells), increased apoptosis in hematopoietic stem/progenitor cells (HSPC) and significant accumulation of G1-phase HSC in the cell cycle analysis, all of which were reminiscent of human myelodysplastic syndromes (MDS). Interestingly, Asxl1G643fs/+ mice became moribund about 600 days after birth when compared to Asxl1+/+ littermates with anemia, leukocytosis, neutrophilia/monocytosis and hepatosplenomegaly, suggesting the development of MDS/myeloproliferative neoplasm (MPN)-like disease, most relevant to human cases with chronic myelomonocytic leukemia.RNA sequencing and gene set enrichment analysis in Asxl1G643fs/+ mice identified derepression of sets of genes that are normally repressed by BMI1, a critical regulator of histone modification in polycomb repressive comprex 1 (PRC1). Among these genes were p16Ink4a, p21, and p15Ink4b all of which were significantly upregulated in Asxl1G643fs/+ LSK cells. We performed chromatin immunoprecipitation (ChIP) assay with an anti-H2AK119ub1 antibody using LSK cells from Asxl1G643fs/+ mice and found that H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1G643fs/+ LSK cells, suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. Co-immunoprecipitation assay and in vitro pull-down assay revealed that the mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild type ASXL1 protein. In addition, we performed ChIP assay with an anti-BMI1 and anti-FLAG antibody using a cell line stably expressed FLAG-tagged wild-type (WT) ASXL1 or mutant form (MT), and found that BMI1 level around the promoter region of p16Ink4a was significantly decreased in the ASXL1 MT-expressed cell line, whereas no significant change in the FLAG level between WT and MT. Furthermore, we intercrossed Asxl1G643fs/+ mice with p16Ink4a heterozygous knock out mice to elucidate the effect of p16Ink4a up-regulation in Asxl1G643fs/+ mice, and found restoration of HSC pool and amelioration of increased apoptosis in HSPC obtained from Asxl1G643fs/+p16Ink4a+/- mice. These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in MDS-like phenotypes represented by decreased HSC pool and aberrant myeloid differentiation in Asxl1G643fs/+ mice.Taken together, our Asxl1 G643fs mutation knock-in mouse line is useful as a model of CHIP or early stage MDS, and our detailed analysis highlights the functional role of ASXL1 mutation on MDS and MDS /MPN development. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau.

A machine learning approach to predicting risk of myelodysplastic syndrome

BackgroundEarly myelodysplastic syndrome (MDS) diagnosis can allow physicians to provide early treatment, which may delay advancement of MDS and improve quality of life. However, MDS often goes unrecognized and is difficult to distinguish from other disorders. We developed a machine learning algorithm for the prediction of MDS one year prior to clinical diagnosis of the disease. MethodsRetrospective analysis was performed on 790,470 patients over the age of 45 seen in the United States between 2007 and 2020. A gradient boosted decision tree model (XGB) was built to predict MDS diagnosis using vital signs, lab results, and demographics from the prior two years of patient data. The XGB model was compared to logistic regression (LR) and artificial neural network (ANN) models. The models did not use blast percentage and cytogenetics information as inputs. Predictions were made one year prior to MDS diagnosis as determined by International Classification of Diseases (ICD) codes, 9th and 10th revisions. Performance was assessed with regard to area under the receiver operating characteristic curve (AUROC). ResultsOn a hold-out test set, the XGB model achieved an AUROC value of 0.87 for prediction of MDS one year prior to diagnosis, with a sensitivity of 0.79 and specificity of 0.80. The XGB model was compared against LR and ANN models, which achieved an AUROC of 0.838 and 0.832, respectively. ConclusionsMachine learning may allow for early MDS diagnosis MDS and more appropriate treatment administration.

PULMONARY ALVEOLAR PROTEINOSIS IN MYELODYSPLASTIC SYNDROME SECONDARY TO GATA-2 MUTATION (MONOMAC SYNDROME)

SESSION TITLE: Medical Student/Resident Chest Infections Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: The majority of the cases of pulmonary alveolar proteinosis (PAP) occur in patients due to autoimmunity with the GM-CSF antibody. We present here a case of a 29-year-old female with GATA-2 mutation (monoMAC) syndrome who presented with dyspnea and was diagnosed with secondary PAP due to myelodysplastic syndrome. CASE PRESENTATION: A 29-year-old female presented to the hospital with the chief complaint of a nonproductive cough for 2 weeks associated with difficulty in breathing with exertion and intermittent fever. She had a history of heterozygous mutation for GATA-2 and was under treatment at a tertiary care center for early Myelodysplastic syndrome secondary to 9q34 deletion. Her medical history was extensive including Mycobacterium avium intracellulare (MAC) Pneumonia and bacteremia diagnosed 8 months before the presentation and was on azithromycin, ethambutol, and rifabutin for it to complete a total of 12 months of treatment. On presentation, she was febrile at 103 degrees Fahrenheit, tachycardiac at 135 beats per minute and saturating 88% on room air. She was placed on 4 L of oxygen by nasal cannula. Labs showed a WBC count of 8.4 10*3/uL (normal 4.0 - 12.0 10*3/uL) with differential showing a low monocyte count at <1 % (normal 2-8 %). Computed Tomography angiogram was obtained which ruled out a pulmonary embolism but showed diffuse and bilateral alveolar and interstitial pulmonary opacification. She had an extensive infectious, autoimmune, metabolic and cardiovascular workup which was all negative. So, bronchoscopy with bronchoalveolar lavage was obtained which showed an exudative effusion with 84 Polymorphonuclear cells, 2 Lymphocytes, 8 Histiocytes, 6 Eosinophils. BAL fluid was negative for respiratory viral PCR, AFB, fungal culture and bacterial culture, galactomannan and Pneumocystis culture. Cytology was also negative for pathogens or malignant cells. Post-Bronchoscopy the patient’s respiratory status improved, and she was weaned off of the oxygen. Pulmonary function tests (PFT) were also obtained there which revealed a non-specific ventilatory defect with decreased Diffusion lung capacity for Carbon monoxide (DLCO). A lung biopsy was obtained there confirming the diagnosis of pulmonary alveolar proteinosis (PAP). The patient had negative anti-GM-CSF antibodies indicating GATA-2 deficiency as the cause of PAP. The patient is scheduled to get a repeat full lung bronchoalveolar lavage and is currently being evaluated for a lung transplant. DISCUSSION: GATA-2 mutation is identified in cases of PAP secondary to hematological malignancies. Whole lung lavage is the standard of care. Treatment of the hematological malignancy may treat the PAP. There is no evidence of corticosteroids in the treatment of secondary PAP. CONCLUSIONS: Clinicians should be mindful of PAP in treating patients with hematological malignancies with respiratory distress. Reference #1: Ishii H, Seymour JF, Tazawa R, Inoue Y, Uchida N, Nishida A, et al. Secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis: a retrospective cohort study in Japan. BMC Pulm Med. 2014;14:37. Reference #2: Seymour JF, Presneill JJ. Pulmonary alveolar proteinosis: progress in the first 44 years. Am J Respir Crit Care Med 2002; 166: 215–235. Reference #3: Milleron BJ, Costabel U, Teschler H, et al. Bronchoalveolar lavage cell data in alveolar proteinosis. Am Rev Respir Dis 1991; 144: 1330–1332. DISCLOSURES: No relevant relationships by Fazal Raziq, source=Web Response No relevant relationships by Muhammad Usama, source=Web Response

Circulating Small Noncoding RNAs Have Specific Expression Patterns in Plasma and Extracellular Vesicles in Myelodysplastic Syndromes and Are Predictive of Patient Outcome.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with large heterogeneity at the clinical and molecular levels. As diagnostic procedures shift from bone marrow biopsies towards less invasive techniques, circulating small noncoding RNAs (sncRNAs) have become of particular interest as potential novel noninvasive biomarkers of the disease. We aimed to characterize the expression profiles of circulating sncRNAs of MDS patients and to search for specific RNAs applicable as potential biomarkers. We performed small RNA-seq in paired samples of total plasma and plasma-derived extracellular vesicles (EVs) obtained from 42 patients and 17 healthy controls and analyzed the data with respect to the stage of the disease, patient survival, response to azacitidine, mutational status, and RNA editing. Significantly higher amounts of RNA material and a striking imbalance in RNA content between plasma and EVs (more than 400 significantly deregulated sncRNAs) were found in MDS patients compared to healthy controls. Moreover, the RNA content of EV cargo was more homogeneous than that of total plasma, and different RNAs were deregulated in these two types of material. Differential expression analyses identified that many hematopoiesis-related miRNAs (e.g., miR-34a, miR-125a, and miR-150) were significantly increased in MDS and that miRNAs clustered on 14q32 were specifically increased in early MDS. Only low numbers of circulating sncRNAs were significantly associated with somatic mutations in the SF3B1 or DNMT3A genes. Survival analysis defined a signature of four sncRNAs (miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p measured in EVs) as the most significantly associated with overall survival (HR = 5.866, p < 0.001). In total plasma, we identified five circulating miRNAs (miR-423-5p, miR-126-3p, miR-151a-3p, miR-125a-5p, and miR-199a-3p) whose combined expression levels could predict the response to azacitidine treatment. In conclusion, our data demonstrate that circulating sncRNAs show specific patterns in MDS and that their expression changes during disease progression, providing a rationale for the potential clinical usefulness of circulating sncRNAs in MDS prognosis. However, monitoring sncRNA levels in total plasma or in the EV fraction does not reflect one another, instead, they seem to represent distinctive snapshots of the disease and the data should be interpreted circumspectly with respect to the type of material analyzed.

Circulating Small Noncoding RNAs As Novel Semi-Invasive Markers of Patient Survival in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with a tendency to transform to acute myeloid leukemia. Several prognostic systems based almost entirely on clinicopathological characteristics have been developed. However, considering large heterogeneity of MDS, there is an ongoing substantial effort to identify new molecular markers, which would improve patient stratification. It has recently been discovered that various types of small noncoding RNAs (sncRNAs) are exported into blood circulation. Encapsulated in extracellular vesicles (EVs) or as a part of various molecular complexes, they play important roles in long distance cell-to-cell communication. Their secretion appears to be controlled and specific and thus may reflect (patho)physiological processes occurring in different cells. As diagnostic procedures move from bone marrow biopsies towards less invasive techniques, circulating sncRNAs have become of particular interest as potential novel semi-invasive biomarkers. Aims: We investigated circulating sncRNAs in MDS on the genome-wide level to better understand MDS pathogenesis and to search specific RNAs applicable as potential biomarkers. We analyzed paired samples from the whole plasma and EV fractions to define which of these two materials is better source of relevant sncRNA-based markers of MDS. Methods: We performed small RNA-seq in paired samples (plasma and EVs) in 42 patients and 17 healthy controls. To associate sncRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Overall survival (OS)‐associated sncRNAs were identified by performing univariate Cox regression along with a permutation test using BRB‐ArrayTools. Multivariate Cox regression analysis with backward variable selection was done to identify independent variables associated with patient survival. Results: Data analysis showed that the most abundant category of RNAs were miRNAs (51 %), followed by rRNAs, piRNAs, tRNAs, and mRNAs. Sample clustering revealed that RNA content of EV cargo is more homogenous than of plasma. Differential analysis identified a striking disbalance in RNA content between plasma and EVs in MDS patients (&gt; 400 significantly deregulated sncRNAs) whereas these sncRNA profiles remained similar in controls. Such disproportion may suggest that mechanisms of sncRNA export into blood circulation may be specifically affected in MDS. As a result, these changes might substantially affect cell-to-cell communication of blood cells, contributing to the disease development. Further, we analyzed levels of individual sncRNAs and found that many hematopoiesis-related miRNAs were significantly increased in MDS patients compared to healthy controls, mostly both in plasma and EVs (e.g., miR-34a-5p, miR-125a-5p, and miR-150). Several miRNAs (e.g., miR-103b, miR-107, and miR-221) showed different levels between early and advanced MDS. Interestingly, the majority of miRNAs from the 14q32 miRNA cluster were specifically increased in early MDS. Additionally, we investigated possible impacts of somatic mutations on expression of circulating sncRNAs. However, only low numbers of circulating sncRNAs significantly associated with mutational status, suggesting no fundamental effects of somatic mutations on sncRNA export from MDS cells. Survival analysis identified sncRNAs with the highest level of association with OS (plasma: miR-1260b, miR-3191-3p, and miR-328-3p; EVs: miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p). To better stratify MDS patients, we defined risk prediction scores (separately for plasma and EVs) that combined effects of the above mentioned sncRNAs. The results showed that EV-combined score significantly increased the predictive power of the survival risk model (Figure 1). Moreover, Cox multivariate analysis revealed that this score is an independent variable the most significantly associated with OS (HR = 2.942, 95% CI 1.786 to 4.849, p &lt; 0.001). Conclusions: Our data demonstrate that profile of circulating sncRNAs is specific in MDS and changes with the disease progression. Monitoring of sncRNA levels in EVs provides better prognostic performance for patient survival (compared to whole plasma), probably due to higher homogeneity of the EV fraction. Supported by grants 16-33617A and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

Predictors of Disease Progression and Survival in Patients with Myelodysplastic Syndrome Secondary to Inherited Bone Marrow Failure Syndromes

Introduction Inherited bone marrow failure syndromes (IBMFSs) are rare genetic disorders characterized by abnormal hematopoiesis resulting in cytopenias and increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Once patients develop MDS the only curative therapy is hematopoietic stem cell transplant (HSCT). The rate of progression from early MDS to advanced MDS and AML is variable and risk factors for progression in IBMFS patients are poorly defined. We hypothesized that certain variables could predict the likelihood of progression from early stages of IBMFS-associated MDS/clonal hematopoiesis to advanced MDS or AML, and that the type of disease progression may impact overall survival (OS). Methods Data were collected from patients prospectively enrolled in the Canadian Inherited Marrow Failure Registry (CIMFR), a collaboration of 1 adult and 16 pediatric hospitals in Canada that care for &gt;95% of pediatric IBMFS patients. IBMFS patients were diagnosed as having a specific syndrome or unclassified IBMFS (UCIBMFS) based on published criteria from our lab and others'. Diagnostic criteria for pediatric MDS defined by Hasle et al. were used. Progression of MDS was defined as 1 or more of: (1) a new cytogenetic abnormality, (2) progression in cytopathology from refractory cytopenia (RC) or refractory cytopenia with ringed sideroblasts (RCRS) to refractory cytopenia with dysplasia (RCD), refractory cytopenia with excess blasts (RCEB) or AML, or (3) increased degree of cytopenia severity. Time to progression was described by Kaplan-Meier analysis and risk factors were evaluated using the Cox proportional hazards model. Results Of 601 patients enrolled in CIMFR, 59 (9.8%) developed cytogenetic clones/MDS. Thirteen (22%) had Fanconi Anemia (FA), 13 (22%) had Shwachman-Diamond Syndrome (SDS), 10 (16.9%) had UCIBMFS, and 23 (39%) had other marrow failure syndromes (i.e. Dyskeratosis Congenita, Severe Congenital Neutropenia, Diamond Blackfan Anemia, GATA2-related disorders). The majority presented with cytogenetic clones/RC (n=45, 76%), 9 (15%) had RCEB, 3 (5%) RCD and 1 (1.7%) RCRS. The most common cytogenetic abnormalities at presentation were -7/-7q (n=18, 30%) and isochromosome 7q10 (n=7, 12%). Four patients had complex cytogenetics (6.8%). Of the patients who developed MDS, 32 (54%) went to HSCT. Patients who developed MDS had significantly worse OS (HR 3, 95% CI 2 to 6, p&lt;0.0001), which varied by IBMFS category. MDS patients with UCIBMFS had a statistically significant lower OS compared to those without MDS (HR 5.7, 95% CI 1.7 to 18.6, p=0.004). In contrast, patients with FA had poor OS regardless of whether or not they developed MDS. Twenty four MDS patients (40%) had disease progression, with a median time to progression of 4.7 months (1.14-131). Nine patients (38%) with disease progression had FA, 5 (21%) had SDS, 4 (17%) had UC, and 6 (25%) had other marrow failure syndromes. Ten patients (42%) developed more advanced cytopathology, 10 (42%) a new cytogenetic abnormality, and 5 (20%) worsening cytopenias. Eight patients progressed from RC to RCEB, with a median time to progression of 5.7 months (1.14 to 113). Progression to more advanced cytopathology was associated with lower OS (HR 2.7, 95% CI 1.0 to 7.4, p=0.046). Median time to progression of cytogenetics was 4.13 months (1.14 to 131), which was not predictive of worse OS (p=0.22). Notably, there was no difference in OS or risk of progression between the -7/-7q and isochromosome 7q groups (p=0.644). Finally, patients who progressed due to worsening cytopenias had significantly lower OS compared to those who did not (p=0.011), but numbers were small. Seventeen (71%) of the MDS patients who progressed underwent HSCT. Conclusion Development of MDS has a significant adverse impact on the OS of IBMFS patients, with disease progression occurring 4.7 months from MDS diagnosis. Progression to advanced cytopathology is associated with decreased survival, while worsening cytogenetic clones and cytopenias may not carry the same risk. Importantly, isochromosome 7q10 is also associated with a risk of progression to more severe hematological disease, and may have an impact on survival. Further analysis of additional variables (i.e. HSCT) will provide insight into the important predictors of survival and disease progression, and help to guide treatment decisions for this high-risk patient population. Disclosures No relevant conflicts of interest to declare.

PS1327 PRO‐NECROPTOTIC RIPK3 AS A STAGE‐DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML

Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

Role of p15(INK4B) Methylation in Patients With Myelodysplastic Syndromes: A Systematic Meta-Analysis

BackgroundTumor suppressor gene cyclin-dependent kinase inhibitor 2B (p15(INK4B)) methylation has been frequently reported in myelodysplastic syndromes (MDS). However, the association between p15(INK4B) methylation and MDS remains elusive. Thus, this meta-analysis was first conducted to evaluate the clinical significance of p15(INK4B) methylation in MDS. Materials and MethodsEligible studies were identified via an online electronic databases search. The overall odds ratios (ORs) or hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. ResultsTwenty-eight studies published between 1997 and 2017 were identified, including 1205 MDS patients and 243 nontumor controls. No evidence of heterogeneity was found in our study. p15(INK4B) methylation was significantly elevated in MDS compared with nontumor controls (OR, 10.37; P < .001). In addition, p15(INK4B) methylation was significantly higher in advanced MDS than in early MDS (OR, 4.70; P < .001) and was linked to an unfavorable overall survival (multivariate analysis: HR, 1.78; 95% CI, 1.23-2.71). Subgroup analyses on the basis of ethnicity and detection method showed that the results remained significant in different subgroups (all Ps < .05). ConclusionOur findings suggest that p15(INK4B) methylation might play an important role in the development, progression, and poor prognosis of MDS. More prospective studies with larger study populations are needed.

Loss-of-Function Mutation in tet2 in Zebrafish Leads to Early MDS like Phenotype

Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia.Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent.We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant.These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss.Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype.Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation.This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

3147 - Loss-of-Function Mutation in TET2 in Zebrafish Leads to Early MDS Like Phenotype

Abstract Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia. Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent. We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant. These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss. Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype. Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation. This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. Disclosures No relevant conflicts of interest to declare.