Disease Progression In Myelodysplastic Syndromes Research Articles

Soluble PD-L1 Predicts Poor Outcome and Disease Progression in Patients with De Novo Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a spectrum of myeloid malignancies typified by regular genetic abnormalities, clonal hematopoiesis, dysfunctional myelopoiesis, inefficient blood cell production, peripheral-blood cytopenia, and an elevated risk for progressing to acute myeloid leukemia (AML). Mounting evidence suggests that the dysregulation of immune checkpoints (ICs) plays critical roles in immune evasion of MDS, which thus led to a series of trials using HMAs in combination with immune checkpoints inhibitors (ICIs). Of these, programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway has been studied most extensively. Recently, forms of cell-free soluble PD-1/PD-L1 (sPD-1/sPD-L1) detection in plasm of patients with solid tumors has open a new paradigm for PD-1/PD-L1 investigations. However, within the context of MDS, the potential dysregulation and prognostic impact of sPD-1/sPD-L1 remains uncertain. This study was aimed to assess roles of sPD-1/sPD-L1 in newly diagnosed MDS patients. Method: Between July 2020 and March 2023, 130 MDS/sAML patients matched the inclusion criteria and 55 healthy individuals with no familial or personal history of autoimmune or cancer disorders were included. The criteria for patient inclusion included: (1) a diagnosis of MDS confirmed pathologically according to 2016/2022 World Health Organization (WHO) classification, or sAML patients with a verified prior MDS history; (2) absence of other conditions like autoimmune diseases or different types of tumors; (3) availability of complete clinical information and survival data. MDS patients were categorized by IPSS-R as: lower-risk (very low risk/ low risk; higher-risk (intermediate risk/high risk/very high risk. Follow-up was conducted until June 16th, 2023. The median follow-up was 14.32 months (range 3.2-34.63 months). Profile of sICs (sPD-L1, sPD-1, sCTLA-4, sGITR, sTIM-3, sOX40, sLAG-3, s4-1BB, sICOS) were tested by ELISA, cytokines (IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α) were detected using Luminex analysis, and the expression levels of PD-1 on T cells (CD3 +, CD4 +, CD8 +, DPT, DNT）were measured using flow cytometry (FCM). Result: In the plasma of 98 newly diagnosed MDS patients, sPD-L1 levels (median 66.80 pg/ml, range 25.00-219.5 pg/ml) were significantly elevated compared to 55 healthy blood donors (median 49.22 pg/ml, range 12.5-218.1 pg/ml). sPD-L1 level was higher in higher-risk IPSS-R groups ( P=0.0380). In addition, higher sPD-L1 levels also showed a correlation with lower hemoglobin concentration, implying a potential link between higher sPD-L1 levels and a more advanced MDS disease state. The AUC for sPD-1 was found to be 0.6333 [95% CI 0.5331-0.7334, P=0.0100] and for sPD-L1, it was 0.6785 [95% CI 0.5836-0.7733, P=0.0002]. The optimal cut-off values were established at 187.5 pg/mL for sPD-1 and 55.08 pg/mL for sPD-L1. Based on these cut-off values, patients with increased sPD-L1 experienced shorter OS ( P=0.0305). By using multivariate Cox model, high expression of sPD-L1 ( P=0.019, HR = 4.172, 95% CI: 1.265-13.755) and high IPSS-R scores ( P=0.030, HR = 2.898, 95% CI: 1.111-7.565) were found to be independent risk factors for newly diagnosed MDS patients. In addition, compared to healthy controls, the PD-1 + T cell subpopulations in MDS patients were significantly reduced, sPD-1 levels were positively correlated with proportion of CD4 + PD-1 + T cells, absolute count of CD4 + PD-1 + T cells and PD-1 + DPT cells, suggesting that CD4 + T cells and DPT cells may be potential sources of sPD-1 in plasma. Subsequently, we analysed the potential roles of cytokines and HMAs in sPD-1/sPD-L1 regulation. It was found that sPD-1 levels had a positive correlation with sPD-L1, IL-2Rα, IP-10, MIG, TNFα, GROα, and TRAIL. sPD-L1 was positively correlated with IL-2Rα, IP-10, MCP-1, MIG, MIP1α, and SCF (Figure 1 A). Notably. We found a reduced sPD-L1 levels in MDS patients who achieved remission after HMA treatment compared to treatment-naïve patients. ( P=0.0302) (Figure 1 B). Conclusions: In conclusion, we found sPD-L1 is an independent risk factors for overall survival in newly diagnosed MDS patients. The elevation of plasma sPD-L1 levels is associated with disease progression in MDS, and may be a potential target for MDS immunotherapy.

Serum Amyloid A1 (SAA1) Secreted By the Stromal Microenvironment Drives Malignant Clonal Proliferation in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Drivers of malignant hematopoietic stem cell clonal proliferation from age-related clonal hematopoiesis (ARCH) to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are not well understood. However, a pro-inflammatory and immune tolerant aging bone marrow (BM) microenvironment is thought to contribute to disease development. Serum Amyloid A1 (SAA1) has been identified as a novel pro-inflammatory oncoprotein the levels of which progressively increase with MDS and AML progression. SAA1 also selectively promotes growth of patient-derived MDS and AML cells independent of cytogenetic or mutational profile. We aimed to characterize the mechanism by which SAA1 drives oncogenesis within the microenvironment. Herein we further stratified the disease marking properties of SAA1 by clarifying the association of its BM levels with disease progression in MDS and AML. SAA1 levels were significantly elevated in BM aspirates from MDS and AML patients as compared to those from healthy age-matched subjects (p=0.0002 and p=0.0052, respectively). The effects of SAA1 on mutated hematopoietic cells were assessed in a model of age-related clonal hematopoiesis (ARCH). Utilizing a CRISPR-Cas9 system, ASXL1-mutant CD34+ hematopoietic stem cells (HSCs) were generated; the replating capacity of both mutated and healthy CD34+ cells were assessed in the presence or absence of SAA1. Notably, SAA1 increased the replating capability assessed by an increase in the number of colonies formed on serial replating of ASXL1-mutant CD34+ cells, but not those of SAA1-treated healthy CD34+ or vehicle treated ASXL1-mutated CD34+ cells, suggesting that the presence of SAA1 favors stemness of mutated HSCs, and implicating SAA1 as a driver of malignant clonality. To investigate the mechanism of action of SAA1 BM mononuclear cells from AML patients or healthy age-matched subjects were treated with SAA1 or vehicle overnight and single-cell RNA sequencing analysis was performed. Seurat and cell cluster annotation analysis showed that AML samples treated with SAA1 had increased numbers of hematopoietic stem and progenitor cells (HSPCs) and plasmacytoid dendritic cells as compared to matched untreated AML samples. Further, Enrichr analysis showed that pathways upregulated by SAA1 in AML samples included pro-inflammatory molecules, anti-apoptotic, and antiviral defense mechanisms (p<0.0001). Shared genes upregulated in these pathways in HSPCs include interferon-induced signals including JAK2, STAT1, and OAS3, and genes implicated in resistance to chemotherapy, including CD44 and CD36, as well as the anti-phagocytosis signal CD47. Lastly, we initiated the development of a blocking monoclonal antibody against SAA1 as a means of inhibiting its potent oncogenic effects on malignant HSCs and examining its potential therapeutic activity. Utilizing a RAW 264.7 NFκB-luciferase reporter cell line, we screened 51 clones for their ability to selectively block SAA1-mediated NFκB activation. 7 optimal candidates were identified which demonstrated dose-dependent reduction in NFκB activity upon SAA1 challenge. Importantly, these candidate clones were specific for SAA1 and did not affect NFκB activation mediated by lipopolysaccharide (LPS)-mediated activation. We observed up to an 85% reduction of SAA1-mediated activity using supernatants from further subcloning of previously selected candidates. Moreover, cell proliferation of a human AML cell line (OCI-AML3) stimulated by SAA1 was markedly reduced in the presence of candidate antibodies, suggesting anti-proliferative activity. Our findings show that SAA1 levels correlates with disease severity in MDS and AML progression, selectively promotes stemness and proliferative potential of ASXL1-mutant cells, and demonstrates unique immune-related mechanisms selecting for malignant clonality. Further development and purification of a novel inhibitory antibody against SAA1 for future in vivo and ex vivo testing is underway as a promising novel target to reduce SAA1-mediated, niche-driven effects with broad implications in the treatment of hematological malignancies.

Establishing a Pathogenic Role for NK Cells As Drivers of Bone Marrow Failure in the Myelodysplastic Syndromes (MDS)

Background: Myelodysplastic syndromes (MDS) are characterized by aberrant bone-marrow (BM) morphology leading to a clonal impairment of hematopoiesis. Worsening BM failure (BMF) and increasing myeloblasts are observed as the predominant consequences of MDS disease progression from very low risk (VLR) to higher risk (HR) to acute myeloid leukemia (AML). Patients are stratified based on the degree of BMF and risk of AML transformation to help guide treatment decisions. Approximately 30% of MDS patients exhibit various autoimmune diseases that can include autoantibodies targeting neutrophils, erythrocytes, and/or platelets. Autoimmune phenomena play an important role in MDS yet are poorly characterized. We hypothesized that early in LR-MDS, dysregulated interactions between stromal cells and developing Natural Killer (NK)/T-cells promote rapid/unregulated development resulting in autoreactivity ( Fig. 1A). We predict dual effector mechanisms: CD16-mediated antibody-dependent cellular cytotoxicity (ADCC) and loss of HLA class I (HLA-I) expression by developing hematopoietic lineage cells making them targets for “missing-self” reactivity ( Fig. 1A). NK cells are important sentinel cells that recognize tumors and dysfunctional cells that lose expression of HLA class I. Such surveillance is guided by interactions between NK inhibitory receptors and HLA class I ligands that ‘train’ NK cells to distinguish diseased cells, exhibiting perturbed HLA expression, from healthy cells with normal expression. The effects of these interactions combined with expression of activating receptors (e.g. CD16, NKG2D, DNAM1, FasL, TRAIL) allow NK cells to engage a target cell through many activating mechanisms, importantly including antibodies eliciting an antibody-dependent cellular cytotoxicity (ADCC) reaction. Autoantibodies are well-established in MDS, yet no one has profiled whether they may trigger ADCC/complement. Further, no one has ever thoroughly characterized BMMCs from LR-MDS patients before. In our study, we observed a clear link between signatures of bone marrow failure at LR-MDS and a significant increase in BM CD57+NK and CD8 T cells expressing a highly activated and dysregulated phenotype. Method: Ex-vivo profiling of 19 MDS patients (N=10 LR/IR-MDS, N=9 HR-MDS) BM mononuclear cells (BMMCs) was performed by mass cytometry (CyTOF)( Fig.1B). Cells were stained with three different antibody panels each consisting of 44 antibodies focused on: specific markers of hematopoiesis and myeloid or lymphoid cell lineages; specific markers of development, effector function, homing, exhaustion, and T-cell memory and proliferation; expression of HLA class I and activating ligands which are critical regulators of NK inhibition. Results: In our study we observed that a major fraction of MDS patients present with clonal-like expansions of BM-derived CD57+CD8+T-cells and natural killer (NK) cells that strongly correlate with autoimmune-cytopenias and BMF-enriched in LR-MDS (Fig.#). We observed that NK-cells and non-classical T-cells were significantly expanded (>2-fold) in LR MDS compared to HR-MDS and healthy-control BM (p<0.01). These non-classical CD57+CD8+T-cells co-expressed HLA-I-reactive and inhibitory NKG2A/KIR receptors as well as activating CD16, NKG2C, NKG2D, NKp46, TRAIL, FasL. In cancers, these cells use innate-like, TCR-independent mechanisms (traditionally ascribed to NK-cells) to target HLA-I-deficient tumor cells. Strikingly, HLA-I was significantly elevated on myeloblasts in LR-MDS compared to HR-MDS (p=0.0007)( Fig.1B), demonstrating NK-evasion strategies, but strongly reduced on dendritic cells, granulocytes, and CD4+T-cells, suggesting susceptible immune targets for destruction by NK-cells (and innate-like CD8+T-cells) (p< 0.0001) ( Fig.1B). BM-derived CD57+NK and CD8+T-cells all co-express very high levels of CD16 making them particularly ADCC reactive. Conclusion: We identified novel innate-like CD8+CD57+ T-cell and NK-cell populations that may play a role in LR-MDS disease pathogenesis. We predict that a strategy to target these cells could help restore hematopoiesis early in the disease state. This may also reduce pressure on myeloblasts to increase HLA-I, permitting improved immune surveillance to control the blast burden and prevention MDS/AML progression. Further experiments are ongoing to validate our observations.

CircZBTB46 predicts poor prognosis and promotes disease progression of myelodysplastic syndromes and acute myeloid leukemia.

Circular RNAs (circRNAs) have been recently identified as important regulators of various diseases, especially cancer. However, the roles of circRNAs in hematologic malignancies have been rarely reported. This study aimed to identify a specific circRNA expression profile in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and to evaluate the biological roles of circRNA in MDS and AML for understanding their clinical significance. Reverse transcription-quantitative PCR was performed to validate the expression of circZBTB46. Kruskal-Wallis test, Kaplan-Meier curves, and the Cox regression model were employed to analyze the clinical significance of circZBTB46. Two specific shRNAs as well as an expression lentiviral vector of circZBTB46 were constructed to identify the biological function of circZBTB46. The impact of circZBTB46 on leukemia cell proliferation, cell cycle distribution, and apoptosis was confirmed using cell viability assay and flow cytometry analysis. The expression of circZBTB46 gradually increased in patients with higher-risk MDS and AML, as compared to controls. CircZBTB46 expression was significantly correlated with important clinical parameters of MDS, including WHO classification, absolute neutrophil count (ANC), marrow blast, IPSS karyotype, IPSS/IPSS-R risk groups, and AML transformation. CircZBTB46 expression was also associated with ANC, marrow blast, cytogenetic risk groups, FLT3-ITD mutation, and treatment response in AML patients. Furthermore, circZBTB46 overexpression was significantly correlated with shorter overall survival (OS, P = 0.0342, median survival time 18.5 vs. 45.4months) and leukemia-free survival (LFS, P = 0.0421) in MDS, also with the shorter OS in AML (P = 0.0293, median survival time 11.6 vs. 16.9months). Functional studies revealed that silencing circZBTB46 expression significantly inhibited proliferation and induced apoptosis in SKM-1, THP-1, and K562 cell lines, while rescue experiments alleviated the siRNA-mediated growth inhibition and apoptosis in these leukemic cells. The present data suggested the essential oncogenic role of circZBTB46, as a progression and survival indicator in both MDS and AML.

Novel RNA epigenetics-driven technologies for rapid prediction of cancer drug resistance.

e19001 Background: The human genome comprises ~99% non-coding DNA sequences (ncDNAs), most of which are actively transcribed into various forms of non-coding RNAs (ncRNAs) that undergo extensive chemical modifications to regulate various functions of the subcellular organelles in human cells ( Statello, et al. Nat Rev Mol Cell Biol 2020). However, the clinical potential of RNA modifications and RNA modifying proteins, i.e., RNA epigenetics, is largely unexplored. In a recent study ( Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct active chromatin structures (ACS) that regulate drug resistance in leukemia cells. This proof-of-concept study enabled us to develop the first-generation of RNA epigenetics-driven drug-predictive system. The primary goal of this study is to validate and optimize this novel drug-predictive system with clinical myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods: We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). Immunohistochemistry (IHC), western blot (WB), co-immunoprecipitation (co-IP), and dot blot were also used in this study. Results: Our data demonstrated significantly increased expression of the NSUN1, NSUN2 and other key components of the drug-resistant ACS in MDS/AML, which is correlated with disease progression and drug resistance in MDS and AML. We have developed novel technologies, i.e., EC- and PL-RCA-coupled CM and FCM (EC-/PL-RCA/CM & FCM) to visualize and quantify RCMTs-mediated ACS and nascent RNA synthesis in MDS/AML. We discovered nascent RNA synthesis as a highly sensitive and super-fast drug-predictive marker that is independent of cell death and apoptosis. Our data demonstrated that PL-RCA/FCM and EC-/CM have a very high sensitivity to detect the NSUN1/2-mediated ACS in leukemia cell lines and clinical MDS/AML specimens. Our preliminary data demonstrated that increased levels of NSUN1/2-mediated ACS detected by PL-RCA/FCM in pre-treatment MDS/AML can rapidly predict the responses to venetoclax and/or azacitidine. We are actively pursuing RNA-seq targeting NSUN1/2-associated nascent RNAs and ACS, which provides a novel strategy to dissect the functional genomics and RNA epigenetics underlying cancer drug resistance. Conclusions: We have developed a novel RCMTs/NSUN1/2-targeting system for rapid prediction and in-depth analysis of drug resistance in MDS/AML. A large sized clinical study is needed to further validate its usefulness in clinical settings.

IDH2 regulates U2AF1 expression and hydroxymethylation in MDS patients

ABSTRACT The expression of some genes regulated by their DNA methylation is involved in pathogenesis and disease progression of myelodysplastic syndrome (MDS), which is characterised by abnormal differentiation and development of myeloid cells. Therefore, it is significant for us to work on investigating what factors regulate U2AF1 expression and hydroxymethylation in MDS patients. However, the members of TET protein family can change 5-methylcytosine (5mC) into 5-hydroxymethylcytosine5-methyl cytosine (5hmC). In general, 5mC and 5hmC levels maintain dynamic equilibrium, and their imbalance is associated with the onset and progression of some tumors. In this study, the expression and 5mC and 5hmC levels of U2AF1 gene decreased significantly after the treatment by decitabine in Mutz-1 cells. The decreased degree of 5hmC is far greater than that of 5mC. IDH2 expression decreased significantly followed by U2AF1 5hmC levels. However, the expression of other hydroxymethylation-related genes such as IDH1, TET1 and TET2 also decreased, but the difference did not achieve significance. Compared with IDH2 or U2AF1 wild-type MDS patients, U2AF1 expression and 5hmC level in patients with these two gene mutations were both significantly reduced.

Pathogenesis and treatment of immune dysregulation associated with myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a clonal disorder based on genomic mutations in hematopoietic stem cells. They are categorized as lower-risk MDS, characterized by peripheral cytopenia; and higher-risk MDS, characterized by progression to acute myeloid leukemia. Previous studies reported that inflammation and immune activation are deeply involved in the pathogenesis of lower-risk MDS. Recent studies elucidated the molecular basis for the activation of inflammatory pathways via dysregulated innate immune system and the resultant cell-death acceleration in lower-risk MDS. Conversely, immunosuppression and immune escape are substantially involved in the pathogenesis and disease progression of higher-risk MDS. VEXAS syndrome is an autoinflammatory disease characterized by clonal hematopoiesis with somatic mutation of UBA1 in hematopoietic stem and progenitor cells and has attracted broad attention as a lower-risk MDS model caused by systemic inflammation. Although therapeutic effects of immunosuppressants are observed for a limited number of patients with lower-risk MDS with inflammation, an optimal treatment should be developed based on their pathology.

High Expression of Plasma IL-1β Levels and Transition of Regulatory T-Cell Subsets Correlate with Disease Progression in Myelodysplastic Syndrome

Objective: Myelodysplastic syndrome (MDS) is a group of heterogeneous hematopoietic clonal diseases. Recent studies have confirmed that there are essential immune differences in the progression of MDS. However, the detailed mechanisms remain unclear. In order to explore the changes of immune disorders during the occurrence and progression of MDS, and identify critical differentially expressed factors as well as prognostic relevance. What's more, to probe the possible immunological mechanism in the progression of MDS. Methods: The study had a prospective design. A total of 86 patients with newly diagnosed MDS were enrolled in this study, where patients were classified as low risk group and high risk group based on the revised international prognostic scoring system (IPSS-R) score. The expression profiles of multiple cytokines in patients’ plasma were determined using Luminex microarray assay, and flow cytometry were used to measure lymphocyte subsets in peripheral blood, especially T regulatory cell (Treg) and its subsets: induced regulatory Treg (iTreg: CD45RO+CD3+CD4+CD25+CD127low+) and naïve regulated Treg (nTreg: CD45RA+CD3+CD4+CD25+CD127low+), and the percentage of Monocytic-myeloid derived suppressor cells (M-MDSCs) in bone marrow. The mRNA expression of NLRP3 inflammasome related molecules was detected by real-time quantitative PCR. P <0 .05 was considered statistically significant. Results: In the high risk group, the plasma levels of some cytokines or chemokines with promoting or amplifying inflammation signals in directly or indirectly way, including IL-9, MIP-1β,GRO-α and TNF-β, and two growth factors influencing the immune microenvironment by regulating the growth and differentiation of corresponding cells, including β-NGF, PDGF-bb, were significantly lower (P<0.05).The expression level of IL-1β was significantly higher in the high risk group (P<0.05) and its expression was significantly correlated with the percentage of M-MDSCs (P<0.0001). What's more, the percentage of bone marrow M-MDSCs facilitated immune tolerance was significantly higher. Although there is no significant difference of Treg between the two groups, both the percentages and absolute counts of nTreg/iTreg was decreased significantly in high-risk group of MDS patients (P<0.05) (Figure). After adjusting the Cox model, nTreg/iTreg was an independent protective factor for OS (P=0.038, HR=0.081, 95% CI: 0.008-0.869), and elevated IL-1β level was an independent prognostic factor for PFS (P=0.018, HR=2.554, 95% CI: 1.175-5.552). Then, we found that the mRNA expression of NLRP3, ASC, Caspase 1 and IL-1β were significantly increased in the high risk group(P<0.05). Conclusion: All above results demonstrated that the expression of pro-inflammatory cytokines and chemokines was increased in low risk MDS, while the amplification of bone marrow M-MDSCs and the transformation of nTreg to iTreg may be the key immune mechanisms leading to disease progression. The activation of NLRP3/IL-1β pathway may play an important role in the progression of disease. The serum levels of IL-1β and the percentage of nTreg /iTreg ratio may be an effective indicator to predict the prognosis of MDS. It provided a basis for clinical prediction and early intervention to improve the prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD123-Directed Bispecific Antibodies for Targeting MDS Clones and Immunosuppressive Myeloid-Derived Suppressor Cells (MDSC) in High-Risk Adult MDS Patients.

There is an urgent need to identify effective strategies to prevent leukemic transformation and induce sustained deep remissions in adult high-risk myelodysplastic syndrome (MDS) patients. This article discusses the clinical impact potential of bispecific antibodies (BiAB) capable of redirecting host T-cell cytotoxicity in an MHC-independent manner to malignant clones as well as immunosuppressive myeloid-derived suppressor cells (MDSC) as a new class of anti-MDS drug candidates. T-cell engaging BiAB targeting the CD123 antigen may help delay disease progression in high-risk adult MDS and potentially reduce the risk of transformation to secondary AML.

U2AF1 mutation promotes tumorigenicity through facilitating autophagy flux mediated by FOXO3a activation in myelodysplastic syndromes

Mutations in the U2 small nuclear RNA auxiliary factor 1 (U2AF1) gene are the common feature of a major subset in myelodysplastic syndromes (MDS). However, the genetic landscape and molecular pathogenesis of oncogenic U2AF1S34F mutation in MDS are not totally understood. We performed comprehensive analysis for prognostic significance of U2AF1 mutations in acute myeloid leukemia (AML) cohort based on The Cancer Genome Atlas (TCGA) database. Functional analysis of U2AF1S34F mutation was performed in vitro. Differentially expressed genes (DEGs) and significantly enriched pathways were identified by RNA sequencing. The forkhead box protein O3a (FOXO3a) was investigated to mediate the function of U2AF1S34F mutation in cell models using lentivirus. Chromatin immunoprecipitation, immunoblotting analyses, and immunofluorescence assays were also conducted. U2AF1 mutations were associated with poor prognosis in MDS and AML samples, which significantly inhibited cell proliferation and induced cellular apoptosis in cell models. Our data identified that U2AF1-mutant cell lines undergo FOXO3a-dependent apoptosis and NLRP3 inflammasome activation, which induces pyroptotic cell death. Particularly, an increase in the level of FOXO3a promoted the progression of MDS in association with restored autophagy program leading to NLRP3 inflammasome activation in response to U2AF1S34F mutation. Based on the result that U2AF1S34F mutation promoted the transcriptional activity of Bim through upregulating FOXO3a with transactivation of cell cycle regulators p21Cip1 and p27Kip1, FOXO3a, a potentially cancer-associated transcription factor, was identified as the key molecule on which these pathways converge. Overall, our studies provide new insights that U2AF1S34F mutation functions the crucial roles in mediating MDS disease progression via FOXO3a activation, and demonstrate novel targets of U2AF1 mutations to the pathogenesis of MDS.

Acquisition Patterns and Interaction of Molecular, Obvious and Cryptic Cytogenetic Abnormalities in Myelodysplastic Syndromes

Abstract Introduction: The acquisition of cytogenetic aberrations (CA) and/or of molecular mutations (clonal evolution, CE) is known to be associated with poor outcome in pts with myelodysplastic syndromes (MDS) (Jabbour et al, Am J Hematol, 2013; Cevik et al, abstract, Onkologie, 2013). The aim of our study was to identify additional cases with CE by taking cryptic CA, which cannot be detected by conventional chromosomal banding analysis (CCB), and their interaction with molecular mutations, into account. Methods: We retrospectively reviewed 103 pts with proven MDS (10x RCUD, 40x RCMD, 10x RAEB-1, 16x RAEB-2, 11x sAML after MDS, 3x MDS-U, 7x CMML, 6x MDS with unknown subtype). Karyotype from CCB and molecular karyotype from SNP-array analysis (SNP-A) were available for all pts. SNP-A and fluorescence in situ hybridization (FISH) analyses were used to detect cryptic CA. For 57 pts results from mutational analysis from a 17-genes Sanger and/or a 54-genes next generation sequencing panel were included. Genetic follow-up and survival data were available for 57 pts, 22 pts were genetically analyzed under treatment with disease modifying therapies. Genetic follow-up comprised analysis of bone marrow and/or frequent sequential genetic monitoring of CD34+ peripheral blood cells. Results: CA were detected at diagnosis in 65/103 pts (63%). By CCB (aberrant in 55/103 pts; 53%) the median number of CA was 1 (range: 0-4). One to 6 (median 1) cryptic CA were detected in 31/103 (30%) pts, 10/31 pts (32%) showed a normal karyotype in CCB. The cryptic CA included cryptic copy number variations (CNV) in 12 pts and CN neutral losses of heterozygosity (CN-LOH) in 19 pts as the sole cryptic CA (14x) or in addition to cryptic CNVs (5x). In 12 pts with CN-LOHs we could identify a homozygous molecular mutation or a cryptic deletion in the region of a CN-LOH. Genes mutated in the region of a CN-LOH were DNMT3A (2p23.3, 2 pts), EZH2 (7q36.1, 2 pts), CBL (11q23.3, 2 pts), RUNX1 (21q22.12, 2 pts), TET2 (4q24), IKZF1 (7p12.2), TP53 (17p13.1), and SRSF2 (17q25.1). The formation of this phenomenon was observed in consecutive samples of 3 pts. From the 57 pts with follow-up data available, 8 showed manifestations of antecedent CE at first diagnosis (2x sub-clones; 6x homozygous mutations in CN-LOHs), from which 7 showed further genetic progression during the course of the disease. CE after first diagnosis was also observed in 20 of the 49 other pts with available follow-up data. The new aberrations of the biggest clone at first CE after diagnosis were partial or complete LOH of chromosome (chr) 7 (6 pts), trisomy 8 (5 pts), chr 21 abnormalities (2 pts), chr 12 abnormalities (2 pts), CN-LOH in 4q, 11p-, 20q-, -X, an additional marker chromosome, tetraploid cells, and mutations in ASXL1 (2 pts), CDKN2A, ETV6, IDH1, and JAK2 . Further steps of CE in these pts are currently being evaluated. Median survival time from detection of the first CE after diagnosis (25 months, 12/20 deaths) did not differ significantly from median survival time from first diagnosis for pts with signs of antecedent CE at first diagnosis (25 months, 5/8 deaths, P=0.92, n.s.). Median survival time of pts without detection of CE was significantly longer (173 months, 8/29 deaths, P=0.005 and Conclusions: In our cohort of MDS pts the frequency of cryptic CA was 30%. A homozygous mutation in a region of CN-LOH, which indicates antecedent CE, was detected in 12% of pts. The presence of such evolutionary patterns at first diagnosis might be indicative of a genetically instable disease as these pts had a shorter survival time and a shorter time to further genetic progression. There is a clear tendency that cytogenetic LOHs preferentially affect regions with molecularly mutated genes, resulting in homozygous mutations. The development of these patterns seems to be an important step towards progression. Our data imply that the relevance of cryptic CA for CE and disease progression in MDS has been underestimated as yet. Cryptic CA should be taken into account for developing prediction models for clinical progress and for improving individualized patients care. Download : Download high-res image (124KB) Download : Download full-size image Disclosures Al-Ali: Gilead: Consultancy, Honoraria; Alexion: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Metz: Celgene: Other: study projects, advanced training support; Novartis: Consultancy, Other: study projects, advanced training support. Germing: Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Platzbecker: Janssen: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Haase: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Gene mutation spectrum of patients with myelodysplastic syndrome and progression to acute myeloid leukemia.

Aim:This study aimed to investigate the regularity of gene mutations in patients with myelodysplastic syndrome (MDS) and in those that progressed to acute myeloid leukemia (MDS/AML).Patients & methods:High-throughput sequencing technology was used to detect gene mutations in 99 newly diagnosed patients with MDS or MDS/AML.Results:Gene mutations were detected in 88 patients. The mutation incidence in the MDS/AML group was significantly higher than that in the MDS group. Statistically significant differences were observed between the MDS with refractory anemia (MDS-RA) and MDS-RA with excess blasts groups and between the MDS/AML and MDS-RA groups.Conclusion:Our data demonstrate that there is a cumulative accumulation of gene mutations, especially in transcription factor genes, during disease progression in MDS and MDS/AML.

Using tissue microarray to detect inflammasome signaling components that contribute to the pathogenesis of myelodysplastic syndrome.

7044 Background: Myelodysplastic syndromes (MDS) are characterized by aberrant maturation, ineffective hematopoiesis, cytopenia, and progression to acute myeloid leukemia. MDS pathogenesis is multifactorial and potentially linked to constitutive innate immune stimulation converging upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. Inflammasome assembly is initiated by both cell-extrinsic stimuli including S100A9, the TLR4 and CD33 ligand, and cell-intrinsic danger signals licensing caspase-1 which activates IL1b and beta-catenin resulting in cell death and cellular proliferation leading to maturation and differentiation blocks. Further, EYA2 has been suggested to be an inflammasome activator, whereas cPLA2 has been suggested to be an inhibitor. The purpose of this study is to determine whether immunohistochemistry (IHC) may be utilized to assess expression of inflammasome components. Methods: An IRB protocol was approved prior to initiating this study. We retrospectively identified 43 low risk MDS patients. A tissue microarray (TMA) was constructed utilizing MDS bone marrow biopsy samples (2-3 representative cores per sample). IL-1, S100A9, EYA2, cPLA2, beta-catenin, and TLR4 expression were assessed by IHC after validation of each antibody. IHC expression was scored independently by two hematopathologists by calculating scores (product of staining intensity x percent expression). IHC expression was compared using Spearman correlation estimate. Demographic and clinical data were collected and correlated with IHC expression using Kruskal-Wallis test, Spearman correlation, and Logrank test. Results: Patients were median 72 years of age, 67% men and included 47% MDS-MLD, 35% MDS-RS, 14% MDS-SLD, 2% MDS del5q and 2% MDS-U. IL-1 expression correlated with beta-catenin expression, r = 0.42, 95% CI 0.115 to 0.658 (p = 0.007). There was a trend towards significance between IL-1 and cPLA2, r = 0.30 (p = 0.067); S100A9 and cPLA2, r = 0.31 (p = 0.052); and S100A9 and EYA2, r = 0.31 (p = 0.057). Percentage EYA2 expression correlated with blast count, r = 0.425 (p = 0.008). The IHC expression of these antigens did not correlate with WHO MDS subclassification, IPSS, R-IPSS, disease progression, or survival (p &gt; 0.05). Conclusions: IHC staining of inflammasome activators using TMA may allow better characterization of molecular pathways contributing the MDS pathogenesis. A correlation was seen between expression of antigens known to be increased downstream of NLRP3 inflammasome activation. Furthermore, increased expression of EYA2 correlated with blast count. A future study will compare expression patterns between normal, low risk MDS and high risk MDS samples and correlate these findings with clinical outcome data to further elucidate the pathogenesis of MDS and identify potential targetable markers for novel therapeutic strategies.

High PRAME expression is associated with poor survival and early disease progression in myelodysplastic syndromes with a low bone marrow blast percentage

We investigated the clinical implications of preferentially expressed antigen in melanoma (PRAME) expression in bone marrow cells of 116 patients with myelodysplastic syndromes (MDS). Quantitative RT-PCR was carried out to examine the PRAME expression level. High PRAME expression was observed in MDS patients classified into higher revised International Prognostic Scoring System (IPSS-R) risk categories (Very high and High) with a high bone marrow blast percentage (5% or higher). Kaplan-Meier analysis demonstrated that high PRAME expression is significantly associated with a poorer overall survival (OS) in MDS patients with a low bone marrow blast percentage (less than 5%) (log-rank test p = .0014) and those classified into lower IPSS-R risk categories (Very Low, Low, and Intermediate) (log-rank test, p = .0035). In contrast, there was no significant association between PRAME expression and OS in MDS patients with a high bone marrow blast percentage or those classified into higher IPSS-R risk categories. In addition, high PRAME expression was associated with early disease progression in MDS patients with a low bone marrow blast percentage. This study suggested PRAME expression to be a prognostic factor in MDS.

Screening for myeloid mutations in patients with myelodysplastic syndromes and AML with myelodysplasia-related changes

IntroductionOne of the most critical complications in myelodysplastic syndromes (MDS) is the progression to acute myeloid leukemia (AML). The dynamics of clonal evolution in MDS and how acquired mutations can be used as biomarkers to track disease progression remains under investigation. Objective and methodHerein, we investigated the frequency of common myeloid clonal mutations (FLT3, NPM1, JAK2, IDH1 and IDH2) in 88 patients with MDS and 35 AML patients with myelodysplasia-related changes, followed at a single reference center in northeastern Brazil. ResultsOverall, 9/88 (10%) of the MDS patients and 9/35 (26%) of the secondary AML patients had at least one mutation. While the JAK2 V617F mutation was the most frequent in the MDS patients, the FLT3, NPM1, IDH1 and IDH2 mutations were more frequently found in the secondary AML group. Furthermore, there was a higher frequency of FLT3, NPM1, IDH1 and IDH2 mutations in MDS patients classified as high-risk subtypes than in those of lower risk. ConclusionDespite the limited sample size, our data suggest that mutations in FLT3, NPM1, IDH1 and IDH2 genes could be potential biomarkers to detect early disease progression in MDS.

MDS-090: Semi-Mechanistic Model to Aid Clinical Understanding of Myelodysplastic Syndromes

Context: Myelodysplastic syndromes (MDS) represent a group of bone marrow disorders involving cytopenia, hypercellular bone marrow, and dysplastic hematopoietic progenitors. Death is commonly caused by the effects of cytopenia, leading to infections or bleeding [1]. Finding efficacious treatment regimens in MDS remains a challenge as it involves understanding of both disease induced and treatment related cytopenias. Objective: The objective was to develop a semi-mechanistic model of disease progression in MDS based on clinical data, involving relevant hematopoietic cells that can describe the effect of drug intervention with venetoclax azacitidine combination therapy. The model was subsequently used to simulate different treatment regimens (with respect to dose, dosing duration in a cycle, and number of cycles) and compare the outcomes. Study Design and Data: Data from an ongoing Phase 1b study evaluating the safety and efficacy of venetoclax (400 mg or 800 mg for 28 days or 100 mg to 400 mg for 14 days in each 28-day cycle) with azacitidine (75 mg/m2 on days 1-7 of each cycle) in treatment-naive patients with higher-risk MDS were used for the analysis. The data contained venetoclax concentrations, neutrophil, red blood cell, and platelet count in the blood and blasts in the bone marrow. Methods: An integrated semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model was developed that accounted for venetoclax PK and azacitidine treatment to describe time dynamics of neutrophils, red blood cells, and platelets. The model also included crowding in the bone marrow due to excess stem cells, MDS blasts, and progenitor cells, and its effect on hematopoiesis. Results: The model described the observed clinical data well and showed predictive ability across considered cell types. Complete remission (CR) and marrow complete remission (mCR) rates were predicted close to observed rates in the study. Simulated efficacy (recovery of blast count, CR, and mCR rates) and safety (neutropenia and thrombocytopenia) endpoints are comparable to the expected outcomes from various dosing regimens. Conclusions: A model describing drug-disease interactions in MDS was developed that integrates multiple end points and differentiates disease driven from drug-induced cytopenia. Reference: [1] Dayyani, F, et al. Cause of death in patients with lower-risk myelodysplastic syndrome. Cancer. 2010 May 1; 116.9:2174-2179. Disclosures: The analysis presented was funded by AbbVie. AbbVie contributed to the design, research, and interpretation of data, writing, reviewing, and approving the publication. NT, JZ, RM, BE, JW, SM, and SG are employees of AbbVie and may hold stock. AHW receives honoraria from Novartis, Astellas, Pfizer, Macrogenics, Abbvie, Genentech, Servier, Celgene, Amgen, Astra Zeneca and Janssen. AHW also receives research funding from Novartis, Celgene, Abbvie, Servier, Astra Zeneca and Amgen. AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax. G-GM is an investigator in AbbVie funded Clinical Trials. DM is a Genentech employee and may own stock. C.M. performed the analysis at AbbVie during an internship. C.M. also receives research funding from an industry consortium (AbbVie Deutschland GmbH & Co. KG, Boehringer Ingelheim Pharma GmbH & Co. KG, Grunenthal GmbH, F. Hoffmann-La Roche Ltd., Merck KGaA and Sanofi) for the PharMetrX program

Next Generation Sequencing and Microrna Assay in a Cohort of Patients Affected By Myelodysplastic Syndromes. an Analysis of Clinical and Genotypic Features

Introduction : The myelodysplastic syndromes (MDS) are a group of clonal haematopoietic stem cell disorders and they can be a consequence of genomic/chromosomal instability. The World Health Organization (WHO) 2008 classification divides the MDS in 4 types: refractory anaemia with excess of blast (RAEB), refractory anaemia (RA), refractory cytopenia with multilineage dysplasia (RCMD) and chronic myelomonocytic leukaemia (CMML). Various mechanisms contribute to the pathogenesis and prognosis of the disease and currently next generation sequencing (NGS) detects pathogenic gene mutations that can allocate patients to different risk classes. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression via post-transcriptional mechanisms and may have oncogenic properties or act as tumour suppressor and have an active role in the onset of myeloid disorders. A prospective study based on integration of NGS and miRNA quantification was launched at Policlinico San Martino in 2017. The aim of this paper is to report the results and the correlation with the clinical features. Patients and methods: 28 consecutive patients were enrolled in this study because they met our eligibility criteria: (i) availability of bone marrow sample at diagnosis, (ii) IPSS and (iii) clinical follow up. Specifically, 11 patients were affected by RAEB, 9 by RA, 5 by RCMD and 3 by CMML. Median age was 66 years old (48 - 85). Eight patients evolved into acute myeloid leukaemia. NGS analysis was performed with Myeloid Oncomine Panel Thermo fisher; miRNA expression was quantified with TaqMan Advanced miRNA Cards. In the context of a machine learning supervised analysis we evaluated if there were significant differences in the miRNAs expression among the patients classified based on the WHO 2008 classification. This analysis was conducted using l1l2-penalized regularization method within PALLADIO, a machine-learning framework that can provide robust variable selection in high-dimensional problems. The identified miRNA signature was characterized though a miRNA enrichment analysis using the webtoolkit WebGestalt. Results: each patient carried a distinctive miRNA signature composed by a median of 650 miRNA. The supervised analysis identified a signature of 24 miRNAs (table 1) able to discriminate the 4 WHO classes with a balanced accuracy of 0.616 (whose significance with respect to the balanced accuracy distribution of the permutation batch experiments has 2.206e-08 p-value). Among these miRNAs, 14 were selected because frequently up-regulated in most the patients of one or more WHO classes (figure 1). NGS identified mutations in 43% of patients affected by RAEB, RCMD and CMML in genes known to be pathogenic for MDS (figure 2). miRNA enrichment analysis allowed the identifications of genes involved in (i) pathways (some in common and some specific for a WHO class) likely involved in the onset of MDS, (ii) in diseases related to MDS and to disease evolution into acute myeloid leukaemia. Some of these genes were the same with the ones identified by NGS in our study population (i.e. TET2, ASLX1, RUNX1, IKZF1, SRSF2) but many others (e.g., GATA2, NfkB, BCL2, CD46, mTOR) were not detected by NGS but are likely to be involved in MDS pathogenesis and disease progression as described previously by other groups (figure 3). C onclusions : our study gives a further input toward the understanding of pathogenesis in MDS through the integration of miRNA analysis with NGS at the diagnosis of MDS. With the identification of specific miRNA signature for each patient it is possible to highlight multiple genes pathway that may contribute to the onset and progression of MDS. In a prospective trial we also aim to verify if a specific miRNA signature is linked to azacitidine resistance. Disclosures Mufti: Celgene: Consultancy, Research Funding.

Identification and validation of SRY-box containing gene family member SOX30 methylation as a prognostic and predictive biomarker in myeloid malignancies

BackgroundMethylation-associated SOX family genes have been proved to be involved in multiple essential processes during carcinogenesis and act as potential biomarkers for cancer diagnosis, staging, prediction of prognosis, and monitoring of response to therapy. Herein, we revealed SOX30 methylation and its clinical implication in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).ResultsIn the discovery stage, we identified that SOX30 methylation, a frequent event in AML, was negatively associated with SOX30 expression and correlated with overall survival (OS) and leukemia-free survival (LFS) in cytogenetically normal AML among SOX family members from The Cancer Genome Atlas (TCGA) datasets. In the validation stage, we verified that SOX30 methylation level was significantly higher in AML even in MDS-derived AML compared to controls, whereas SOX30 hypermethylation was not a frequent event in MDS. SOX30 methylation was inversely correlated with SOX30 expression in AML patients. Survival analysis showed that SOX30 hypermethylation was negatively associated with complete remission (CR), OS, and LFS in AML, where it only affected LFS in MDS. Notably, among MDS/AML paired patients, SOX30 methylation level was significantly increased in AML stage than in MDS stage. In addition, SOX30 methylation was found to be significantly decreased in AML achieved CR when compared to diagnosis time and markedly increased in relapsed AML when compared to the CR population.ConclusionsOur findings revealed that SOX30 methylation was associated with disease progression in MDS and acted as an independent prognostic and predictive biomarker in AML.

Selective inhibition of BCL-2 is a promising target in patients with high-risk myelodysplastic syndromes and adverse mutational profile.

Somatic mutations in genes such as ASXL1, RUNX1, TP53 or EZH2 adversely affect the outcome of patients with myelodysplastic syndromes (MDS). Since selective BCL-2 inhibition is a promising treatment strategy in hematologic malignancies, we tested the therapeutic impact of ABT-199 on MDS patient samples bearing an adverse mutational profile. By gene expression, we found that the level of pro-apoptotic BIM significantly decreased during MDS disease progression in line with an acquired resistance to cell death. Supporting the potential for ABT-199 treatment in MDS, high-risk MDS patient samples specifically underwent cell death in response to ABT-199 even when harbouring mutations in ASXL1, RUNX1, TP53 or EZH2. ABT-199 effectively targeted the stem- and progenitor compartment in advanced MDS harbouring mutations in ASXL1, RUNX1, TP53 or EZH2 and even proved effective in patients harbouring more than one of the defined high-risk mutations. Moreover, we utilized the protein abundance of BCL-2 family members in primary patient samples using flow cytometry as a biomarker to predict ABT-199 treatment response. Our data demonstrate that ABT-199 effectively induces apoptosis in progenitors of high-risk MDS/sAML despite the presence of adverse genetic mutations supporting the notion that pro-apoptotic intervention will hold broad therapeutic potential in high-risk MDS patients with poor prognosis.

Functional expression of Tim-3 on blasts and clinical impact of its ligand galectin-9 in myelodysplastic syndromes

T-cell immunoglobulin mucin-3 (Tim-3), an inhibitory immune checkpoint receptor, is highly expressed on acute myeloid leukemia cells and its ligand galectin-9 is reported to drive leukemic progression by binding with Tim-3. However, it remains unclear whether the Tim-3–galectin-9 pathway is associated with the pathophysiology of myelodysplastic syndromes (MDS). Thus, we investigated the expression and function of Tim-3 and the clinical impact of its ligand galectin-9 in MDS. Tim-3 expression levels on MDS blasts by CD45/side-scatter or CD34/CD45 gating were increased as MDS progressed to the advanced stage. Tim-3 expression in the MDS blasts was upregulated in the presence of the cell culture supernatant of human stromal cells or the MDS-related cytokine transforming growth factor-β1. The proliferation of Tim-3+ MDS blasts was inhibited by the blockade of anti-Tim-3 antibody. Furthermore, plasma levels of galectin-9 were elevated as MDS progressed to the advanced stage in 70 MDS/acute leukemia transformed from MDS patients and was a prognostic factor in 40 MDS patients. Our data demonstrated that the Tim-3-galectin-9 pathway is associated with the pathogenesis and disease progression of MDS. These findings provide new insight into potential immunotherapy targeting the galectin-9–Tim-3 pathway in MDS.