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Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly originate from mutations in the splicing factorSF3B1(SF3B1mt).SF3B1mtcause RNA mis-splicing, mechanistically established as the major driver of RS development. However, little is known about RS fate and biology after their initial formation in the human bone marrow. We here achieve isolation of viable RS from patient samples, enabling the first complete investigation ofSF3B1mtdevelopment from stem cell to RS. We show that RS skew MACS-isolated CD34+data towards erythroid features not recapitulated in single-cell RNAseq. We demonstrate that RS divide, differentiate, enucleate and actively respond to mis-splicing/oxidative stress, decreasing wildtype stem cell fitness via GDF15 overproduction. We identify circulating RS as a uniform clinical feature associated with disease burden. Finally, we establish thatSF3B1mtmis-splicing intensifies during erythroid differentiation and demonstrate through combined transcriptomics/proteomics an uncoupling of RNA/protein biology in RS encompassing severe and dysfunctional mis-splicing of proapoptotic genes.Statement of significanceWe here combine a novel method for RS isolation with state-of-the-art multiomics to perform the first complete investigation ofSF3B1mtMDS-RS hematopoiesis from stem cell to RS. We identify the survival mechanisms underlyingSF3B1mterythropoiesis and establish an active role for erythroid differentiation and RS themselves inSF3B1mtMDS-RS pathogenesis.

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Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader ZBTB33 as well as in YLPM1, SRCAP, and ZNF318. We also identified these mutations at low frequency in patients with myelodysplastic syndrome. Zbtb33-edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage in vivo and increased genome-wide intron retention. ZBTB33 mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and myelodysplastic syndromes.Significance:Mutations in known driver genes can be found in only about half of individuals with clonal hematopoiesis. Here, we performed a somatic mutation discovery effort in nonmalignant blood samples, which identified novel candidate genes that may play biological roles in hematopoietic stem cell expansion and hematologic malignancies.This article is highlighted in the In This Issue feature, p. 403

The incidence and prognosis of clonal hematopoiesis in patients with isolated neutropenia among patients with idiopathic cytopenia of undetermined significance (ICUS), known as ICUS-N or chronic idiopathic neutropenia (CIN) patients, is poorly defined. The current study sought to investigate the frequency and clinical significance of mutations of genes implicated in myeloid malignancies using next-generation sequencing in patients with CIN (n = 185) with a long follow-up. We found that 21 (11.35%) of 185 patients carried a total of 25 somatic mutations in 6 genes with a median variant allele frequency of 12.75%. The most frequently mutated genes were DNMT3A and TET2 involving >80% of patients, followed by IDH1/2, SRSF2, and ZRSR2. The frequency of transformation to a myeloid malignancy was low in the total group of patients (5 of 185 patients [2.70%]). However, from the transformed patients, 4 belonged to the clonal group (4 of 21 [19.05%]) and 1 to the nonclonal group (1 of 164 [0.61%]), indicating that the presence of mutation(s) confers a relative risk for transformation of 31.24 (P = .0017). The variant allele frequency of the mutant clones in the transformed patients was >10% in all cases, and the genes most frequently associated with malignant transformation were SRSF2 and IDH1. No significant differences were identified between the clonal and nonclonal groups in the severity of neutropenia. Patients with clonal disease were older compared with nonclonal patients. These data contribute to the better understanding of the heterogeneous entities underlying ICUS and highlight the importance of mutation analysis for the diagnosis and prognosis of patients with unexplained neutropenias.

Large scale high-throughput DNA sequencing studies have identified clonal hematopoiesis (CH) as a clinical phenomenon characterized by a disproportionately large clonal population in the hematopoietic system with a shared mutational background. CH originates through mutations in hematopoietic stem and progenitor cells (HSPCs) which provide a proliferative advantage over unmutated HSPCs and has been characterized as a risk factor for myeloid neoplasm (MN) development. Large population studies found that CH is an age-related event which is commonly found in association with milder phenotypes such as cytopenia, mild monocytosis, intravascular hemolysis, or chronic inflammation. More importantly, the vast majority of individuals with CH are asymptomatic and healthy people of advanced age, where the impact of CH is thus considered to be of indeterminate potential (CHIP). These conditions are sometimes referred to as benign to facilitate distinction from overt MN but, despite this definition, may still result in severe illness, reduced overall survival, and increased risk of hematologic neoplasms development and all-cause mortality. The purpose of this review is to describe clinical conditions associated with CH, the clinical significance of CH-related clinical phenotypes, and the determinants of progression from CH to overt MN following the paradigmatic example of SF3B1-driven CH.

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m 6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2P95 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2P95-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.

Purpose of review Clinical and experimental studies have uncovered relevant clinical implications of clonal hematopoiesis. However, the true magnitude of this process, clonal dynamics over time and mechanisms of progression into overt malignancy remain to be largely elucidated. In this article, the consequences of clonal hematopoiesis, its significance in the context of cytopenia, and its implications in the clinical management of patients with myeloid malignancies are reviewed and discussed. Recent findings Clonal hematopoiesis has been associated with higher risk of hematologic cancers, as well as of death from cardiovascular causes. Clonal hematopoiesis has been proven clinically relevant in the context of disorders characterized by peripheral blood cytopenia, including aplastic anemia, cytopenia of undetermined significance, as well as unexplained anemia of the elderly. Summary The available evidence has been proving the utility of somatic mutational analysis in patients with unexplained cytopenia, as well as in those receiving a diagnosis of myeloid neoplasm, enabling more accurate diagnosis, risk assessment, effective therapeutic strategies and residual disease monitoring. The access to a minimally invasive assessment is paving the way for screening programs of clonal hematopoiesis in individuals with absent or mild hematologic phenotype, as well as for therapeutic targeting of preleukemia cells.

Understanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggests a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows to infer conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology are a steppingstone towards a comprehensive cell atlas of the BM microenvironment.

Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader, ZBTB33, as well as in YLPM1, SRCAP, and ZNF318. We also identified these mutations at low frequency in myelodysplastic syndrome patients. Zbtb33 edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage in vivo and increased genome-wide intron retention. ZBTB33 mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and MDS.

Diagnostic criteria for hypoplastic myelodysplasic syndrome (h-MDS) have not been clearly established, making the differential diagnosis from other bone marrow failure syndromes (BMF) challenging. In this study, we aimed to delineate clinical, histopathological, and molecular features of h-MDS, based on a large and well-annotated cohort of patients with bone marrow (BM) hypocellularity. The study included 534 consecutive adult patients with hypocellular BM (278 h-MDS and 136 aplastic anemia), and 727 with normo- or hypercellular MDS (n-MDS). Comparison of clinical features of patients with h-MDS as defined by BM cellularity ≤25% (n = 204) or reduced age-adjusted cellularity (n = 74) did not reveal significant differences. We developed a diagnostic score to discriminate h-MDS from non-malignant BMF based on histological and cytological variables with the highest specificity for MDS (h-score). The information from chromosomal abnormalities and somatic mutation patterns was then integrated into a cyto-histological/genetic score (hg-score). This score was able to segregate two groups of h-MDS with a significantly different risk of blast progression (P

Clonal cytopenia of undetermined significance (CCUS) is associated with an increased risk of developing a myeloid neoplasm with myelodysplasia (MN). To identify the features of the mutant clone(s) that is associated with clinical phenotype and progression, we studied the following cohorts of individuals: 311 patients with idiopathic cytopenia of undetermined significance (ICUS), 532 community-dwelling individuals without hematologic phenotype (n = 355) or with unexplained anemia (n = 177), and 592 patients with overt MN. Ninety-two of 311 (30%) patients with ICUS carried a somatic genetic lesion that signaled CCUS. Clonal hematopoiesis (CH) was detected in 19.7% and 27.7% of nonanemic and anemic community-dwelling individuals, respectively. Different mutation patterns and variant allele frequencies (VAFs) (clone metrics parameters) were observed in the conditions studied. Recurrent mutation patterns exhibited different VAFs associated with marrow dysplasia (0.17-0.48), indicating variable clinical expressivity of mutant clones. Unsupervised clustering analysis based on mutation profiles identified 2 major clusters, characterized by isolated DNMT3A mutations (CH-like cluster) or combinatorial mutation patterns (MN-like cluster), and showing different overall survival (HR, 1.8). In patients with CCUS, the 2 clusters had different risk of progression to MN (HR, 2.7). Within the MN-like cluster, distinct subsets with different risk of progression to MN were identified based on clone metrics. These findings unveil marked variability in the clinical expressivity of myeloid driver genes and underline the limitations of morphologic dysplasia for clinical staging of mutant hematopoietic clones. Clone metrics appears to be critical for informing clinical decision-making in patients with clonal cytopenia.

Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5′ terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5′ untranslated region (UTR), including 5′ terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5′ PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).

SF3B1-mutant myelodysplastic syndrome (MDS) has recently been proposed as a distinct disorder characterized by ring sideroblasts, ineffective erythropoiesis and good prognosis. Selected co-occurring genetic abnormalities were reported associated with significantly worse outcome and suggested as exclusion criteria for the proposed entity. However, it remains unclear how a limited spectrum of co-occurring drivers affects SF3B1-mutant MDS biology to determine evolution from a relatively indolent condition to high risk malignancy. To gain a better insight into the clonal progression of SF3B1-mutant MDS, we analyzed SF3B1 co-mutations in a cohort of 176 SF3B1-mutated patients diagnosed with a myeloid neoplasm. RUNX1 and STAG2 were the only co-mutated genes found significantly associated with advanced disease phenotype (i.e. MDS with excess blasts and secondary acute myeloid leukemia) (OR=18.36 (2.18-862.91), P=0.001, and OR= Inf (3.57-Inf), P RUNX1 and STAG2 control hematopoietic stem and progenitor cell self-renewal and differentiation by regulation of gene expression. To explore the biological impact of RUNX1 or STAG2 loss in the context of SF3B1-mutant MDS, we disrupted RUNX1 or STAG2 using CRISPR/Cas9 in an induced pluripotent stem cell (iPSC) model of MDS-RS that we previously established by reprogramming of bone marrow cells from SF3B1-mutant individuals. This patient-derived system allows hematopoietic progenitor expansion through doxycycline-mediated expression of 5 transcription factors (5F-HPC) and multilineage differentiation upon doxycycline withdrawal. We asked how RUNX1 or STAG2 disruption affected SF3B1-mutant HPCs self-renewal and differentiation in our model of MDS-RS, and sought to define the underlying transcriptional changes. RUNX1-edited cells maintained significantly higher proportion of CD34 + HPCs, suggesting that RUNX1 mutation increases self-renewal capacity of SF3B1-mutant progenitors. Consistently, SF3B1/RUNX1 double-mutant 5F-HPCs showed positive enrichment of HSC-specific gene signatures. This was accompanied by broad upregulation of inflammatory programs, recapitulating the activated gene signatures we identified in SF3B1/RUNX1 co-mutated patients in our cohort. RUNX1 disruption promoted myeloid skewing at the expense of erythroid differentiation in SF3B1-mutant cells. Consistent with this, granulocyte-monocyte progenitors (GMP) and myelo-erythroid transcriptional programs were positively and negatively enriched, respectively. By contrast, in SF3B1/STAG2 double-mutant 5F-HPCs, both erythroid and myeloid populations were reduced compared to SF3B1 single mutant controls, suggesting a block in both myeloid and erythroid differentiation, despite the presence of pro-differentiation signals. This was supported by a profound down-regulation of genes involved in the response to external stimulus and suppression of GMP specific transcriptional signature. In summary, we identified RUNX1 and STAG2 mutations as main drivers of disease progression in SF3B1-mutant patients, and generated extensive cell line panels to interrogate their functional interaction with mutant SF3B1. By applying CRISPR/Cas9 editing to our MDS-RS model, we could overcome limited availability of primary MDS samples to show that RUNX1 or STAG2 co-mutations drive progression through distinct biological mechanisms in SF3B1-mutant HPCs. Disclosures No relevant conflicts of interest to declare.

Understanding the regulation of normal and malignant human hematopoiesis requires a comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Recent studies using scRNA-seq technologies have shed light on the organization of the hematopoietic regulatory microenvironment in the mouse. These studies have resolved some of the controversies regarding the overlap of stromal populations, the description of certain discrete stromal cells as professional, hematopoietic cytokine-producing populations, but also helped to delineate the relationship between specific stromal cell types in the murine BM. Nevertheless, these studies are limited by the number of cells sequenced, potentially hampering our ability to resolve the full spectrum of cellular states and differentiation stages that define the stromal BM microenvironment. Further, knowledge on the conservation of the cellular composition in the human BM stroma is in its infancy due to the difficulty of obtaining high-quality samples with sufficient stromal cell numbers from healthy individuals. This leaves us with two outstanding challenges; how to piece together such different fragments towards a comprehensive molecular atlas and to what extent such an atlas in mice is conserved in the human bone marrow. Here, we dissect the intrinsic organization and the heterogeneity within the endothelial (EC) and mesenchymal cell populations (MSC) governing the BM microenvironment in mouse and human. This was accomplished through customized bioinformatics integration of multiple scRNA-seq datasets along with the inclusion of over 50.000 murine and human bone marrow stromal cells. By these means, we were able to identify new subsets of MSC and EC, but more importantly, to define new molecular markers to identify highly specialized subpopulations of cells in the murine BM microenvironment. Pathway enrichment analysis unveiled multiple, potentially transient cell states defined by differential gene expression and the enrichment of specific functional characteristics. Importantly, 14 EC subsets were characterized by enrichment in pathways known to be essential for endothelial homeostasis maintenance, demonstrating a high degree of specialization in the endothelium. Similarly, 11 transient cell states in the MSC compartment were defined and characterized by their differentiation capacity. Importantly, our deep deconvolution of the heterogeneous mesenchymal and endothelial compartments became feasible only by integrating multiple datasets. Furthermore, based on the knowledge generated in the mouse, we were able to describe how much of the information and targets from the mouse can be of interest in human characterization. This analysis identified the expression of the human orthologs to the murine cluster-defining genes with different degrees of enrichment in the endothelium and mesenchyme. Moreover, some of these shared genes in mice and human stromal cells corresponded to the GO-defining genes of the different clusters identified in the mouse. These findings suggest a significant degree of conservation regarding the cellular states that define the stromal microenvironment in mouse and human. Although additional studies and improved processing of human samples will be required for deep characterization of the human BM microenvironment, these preliminary results validate our integrative cross-species approach. Taken together, our study provides a deeper understanding of the composition and specialization of the BM microenvironment and point towards a significant degree of conservation between species. Moreover, we demonstrate the usefulness of the multi-dataset integration and the customized clustering approach used in our study to improve the resolution of complex tissues and organs. This approach promises to aid in the construction of cell atlases by reducing the resources associated with sequencing that a single lab will need to invest in order to obtain meaningful depth in single-cell analysis. Future studies integrating genome, transcriptome, epigenome, proteome, and anatomical positioning together with functional assays to correlate descriptive phenotypes with functional data will help fully resolve the composition, regulation, and connectivity in the BM microenvironment in health and disease. Figure 1 Figure 1. Disclosures Paiva: Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. Saez: Magenta Therapeutics: Patents & Royalties. Prosper: BMS-Celgene: Honoraria, Research Funding; Janssen: Honoraria; Oryzon: Honoraria.

Background and aims - The 2016 revised WHO classification incorporated somatic mutation in SF3B1 spliceosome gene within the diagnostic criteria of myelodysplastic syndrome (MDS) with ring sideroblasts (RS). However, SF3B1wt MDS-RS display significantly different clinical features and outcome from those of SF3B1mut MDS-RS. Recently, the recognition of SF3B1-mutant MDS as a distinct nosologic entity has been proposed to overcome this limitation. Methods - To evaluate the biological relevance of this proposal, we studied a consecutive cohort of 132 MDS with RS >5% using a pangenomic approach (targeted-DNA sequencing, genome-wide copy number variation analysis and bulk RNA-sequencing of CD34+ bone marrow mononuclear cells). 16 age-matched healthy individuals and 43 MDS-SLD/MLD negative for both splicing mutation and RS were included in this study as controls. Results - Unsupervised clustering analysis based on mutation profiles identified two major clusters predicted by SF3B1 mutation (87 MDS-RS-SF3B1mut and 45 MDS-RS-SF3B1wt). The most recurrently mutated genes in MDS-RS-SF3B1wt were TP53(40%), SRSF2(38%), TET2 (33%), ASXL1 (21%) and DNMT3A (12%). SRSF2 and TP53 mutations were found to be mutually exclusive with SF3B1 (p-value Differential gene expression analysis results were incorporated into a specific expression signature highly predictive of MDS-RS-SF3B1 mutand MDS-RS-SF3B1wt subgroups (Figure 1). The resulting gene clusters were classified in RS-specific genes (cluster 1 and 2) and SF3B1-specific genes (cluster 3 and 4). RS-specific genes comprising heme and hypoxia genes were enriched (Figure 2AB) and correlated with RS percentage (p Conclusions - This study contributes to unveil molecular features of SF3B1-mutant MDS and provides further evidence to support recognition of somatic SF3B1 mutation as a disease-defining genetic lesion. Disclosures Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by aberrant HSC differentiation, cytopenia, and an increased risk of progression to leukemia. The low-risk subtype MDS with ring sideroblasts (MDS-RS) is specifically characterized by expanded and ineffective erythropoiesis, with more than 80% of patients displaying mutations in the core spliceosome component SF3B1 (SF3B1 mt). A hallmark of the MDS-RS bone marrow (BM) is the progressive accumulation of ring sideroblasts (RS), erythroblasts displaying perinuclear mitochondria loaded with aberrant ferritin-iron complexes. Whilst several in vitro and in vivo model systems exist for studying the impact of SF3B1 mt on erythropoiesis and RS development, primary SF3B1 mt erythroid biology remains largely unexplored due to the inability to purify live SF3B1 mt cells or fully replicate BM conditions in vitro. To address this issue, we developed an innovative two-step method to isolate live ring sideroblasts from SF3B1 mt MDS-RS BM aspiration material with extremely high purity (as determined through droplet digital PCR-based genotyping [Fig. 1A] and morphology-based detection through Prussian blue staining [Fig. 1B,C]). Unexpectedly, evaluation of matching peripheral blood samples showed that circulating ring sideroblasts are strikingly common in MDS-RS (Fig. 1D), with their abundance being significantly positively associated with clinically-determined BM RS frequencies and serum erythropoietin levels, as well as negatively associated with hemoglobin levels. Through high-throughput Chromium 3'-based single-cell RNA sequencing (scRNAseq) analysis of purified RS, we then showed that these cells comprise a heterogeneous population encompassing all stages of the erythroid differentiation continuum, from early progenitors to orthochromatic erythroblasts (Fig. 1E). The RS transcriptome was shown to be dynamically regulated towards the maintenance of cell survival during late terminal erythroid differentiation (exemplified through parkin 1 [PINK1] expression), with SF3B1 K700E erythroblasts employing multiple strategies to preserve homeostasis despite undergoing extreme oxidative stress. These observations were confirmed through a parallel whole-transcript RNAseq investigation comprising CD34 + and GPA +-enriched samples obtained from normal bone marrow (NBM) donors and SF3B1 K700E MDS-RS patients, as well as purified RS samples. This bulk RNAseq experiment validated the RS transcriptomic signature observed in scRNAseq (Fig. 1F) and allowed for a detailed investigation of RNA splicing. SF3B1 K700E-associated alternative splicing in CD34 + and RS was consistent with previous literature, but also highly context-dependent and with substantial changes in scope and magnitude throughout erythroid differentiation (Fig. 1G-I). Finally, we substantiated these RNAseq results through Tandem Mass Tag-based semi-quantitative proteomic analysis of purified RS and GPA-enriched cells from NBM donors and MDS-RS patients. We confirmed that ring sideroblast survival is heavily dependent on redox balance modulation and suppression of ER stress via an increased dependence on glutamine, mirroring the molecular mechanisms observed in malignancy. Additionally, our data strongly indicate that the RS population is a major modulator of the MDS-RS BM microenvironment due to expression of stress factors (with particular emphasis on GDF15, erythroferrone and IL-18). In conclusion, our integrative analysis of primary RS constitutes a unique platform for the study of MDS-RS, with special interest for the investigation of potential drivers of disease severity or treatment avenues. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment.

Therapy-related myeloid neoplasms, such as myelodysplastic syndromes (t-MDSs) and acute myeloid leukemia (t-AML), account for 10% to 20% of all myeloid neoplasms and are recognized as a distinct subgroup according to 2008 World Health Organization (WHO) criteria.[1][1] Although t-MDS/t-AML are often

Chronic lymphocytic leukemia (CLL) and myeloproliferative neoplasms (MPN) may occur concomitantly. However, little is known about the pathobiological characteristics and interaction between the neoplastic clones in these rare cases of coinciding malignancies. We retrospectively examined the clinical and biological characteristics of 13 patients with concomitant CLL and MPN--eight primary myelofibrosis (PMF), three essential thrombocytosis (ET), and two polycythemia vera (PV)--who presented to our institution between 1998 and 2014, and tested all patients for MPN-specific aberrations, such as JAK2, MPL and CALR mutations. Along with epidemiological and molecular characterization of this rare condition, we found that JAK2 mutation can be detected 9 years prior to PMF diagnosis, suggesting that PMF clinical phenotype may require several years to develop and CLL/MPN clinical co-occurrence might be sustained by common molecular events. Some features of these patients suggest that pathobiologies of these diseases might be intertwined.

Splicing factors are the most frequently mutated genes in myeloid neoplasms with myelodysplasia, including myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and acute myeloid leukemia with myelodysplasia-related changes (AML-MDS). SF3B1 mutations was shown to be strongly associated with disease phenotype with ring sideroblasts, and SF3B1-mutant MDS has been proposed as a distinct disease subtype (Malcovati et al. Blood 2015). Conversely, SRSF2 has been found to be mutated in chronic myelomonocytic leukemia (CMML), and to be associated with multilineage dysplasia and excess blasts in MDS, as well as with clinico-pathological features of secondary AML (Linsdley et al. Blood 2015). In this study, we carried out a comprehensive mutation analysis in a well-annotated prospective cohort of 551 patients with myeloid neoplasms with myelodysplasia, and focused on clinical and molecular features of SRSF2-mutated patients with the aim to characterize patterns of co-occurring mutations and to identify genotype-phenotype correlations. A core panel of 54 genes recurrently mutated in myeloid neoplasms was analyzed using an Illumina HiSeq (Illumina Inc., CA, USA). Multivariable survival analyses were performed by means of Cox proportional hazards regression accounting for left censoring of the observation at the time of mutation assessment. Unsupervised hierarchical clustering analyses were performed in order to identify homogeneous groups of patients with respect to the variables of interest. Overall, 102 patients carrying SRSF2-mutation were identified, including 37 MDS (2 MDS with single lineage dysplasia, 9 with multilineage dysplasia, 6 with ring sideroblasts, 20 with excess blasts), 40 MDS/MPN (35 CMML, 5 MDS/MPN unclassified) and 25 AML-MDS. The median number of co-mutated genes was 2 (range 0 to 5); SRSF2 mutation was the unique mutation detected in 11 cases (5 AML-MDS, 4 MDS/MPN and 3 MDS). The most frequently co-mutated genes were TET2 (31/102 cases, 30%), ASXL1 (28%), IDH1/IDH2 (24%), RUNX1 (21%), STAG2 (14%) and CBL (10%). A significant association was found between co-mutation of TET2 and SRSF2 and myeloid neoplasm with monocytosis (P Then, we carried out uni- and multivariable Cox-regression analyses, and found that SRSF2-mutant MDS/MPN showed a significantly better prognosis compared to MDS (HR=.44, P=.008) and AML-MDS (HR=.47, P Finally, we performed unsupervised hierarchical clustering analyses including current WHO classification criteria and gene mutations. This analysis identified one cluster including patients with MDS/MPN according to current WHO criteria, and a second cluster comprising patients with MDS or AML-MDS, in which a distinct subgroup including SRSF2-mutated patients was identified, irrespective of WHO bone marrow blast cut-offs. In conclusion, these results suggest that SRSF2 mutation is able to identify a distinct subset of myeloid neoplasms at the boundaries of MDS and AML, irrespective of current WHO classification criteria. Conversely, SRSF2-mutant MDS/MPN show different co-mutation patterns and clinical outcomes, supporting a classification in a separate category according to current WHO criteria. The recognition of a distinct entity with a unique molecular basis may significantly improve the diagnosis, classification and treatment of patients with these disorders. Disclosures No relevant conflicts of interest to declare.

The diagnostic approach to unexplained cytopenia is hampered by the poor specificity of dysplastic changes that may complicate the distinction between myeloid neoplasms (MN) and non-malignant cytopenias. In the last years, several somatic mutations were identified in MN; however, the diagnostic value of mutation analysis needs to be defined. In this study, we performed a mutation screening in a prospective cohort of patients with unexplained cytopenia undergoing a comprehensive diagnostic work-up, with the aim to estimate the predictive value of somatic mutations. This study included two cohorts: a learning cohort that consisted of 683 consecutive patients investigated for unexplained cytopenia at the Policlinico San Matteo & University of Pavia, Italy, between 2003 and 2015; and a validation cohort, including 190 patients referred as second opinion for suspected MDS. A set of 42 genes was analyzed on DNA from peripheral blood granulocytes using Illumina HiSeq (Illumina Inc., CA, USA). The diagnosis of patients in the learning cohort was MN in 409 cases (233 MDS, 86 MDS/MPN, 35 MPN; 55 AML), other cytopenia in 120 cases, whereas in 154 patients a provisional diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) was adopted. After a median follow-up of 22 months (range 3-136), 38 patients in this category developed a MN (ICUS-MN). The most frequently mutated genes were TET2 (171/683, 25%), ASXL1 (15%), SRSF2 (14%), SF3B1 (11%), DNMT3A (10%), RUNX1 (9%). Significantly higher number of mutations per subject and variant allele frequency (VAF) were observed in MN (n=2, range 0-9; VAF=0.39, 0.03-0.57) compared with ICUS (n=0, 0-7; VAF=0.31, 0.03-0.51) or other cytopenia (n=0, 0-2; VAF=0.06, 0.03-0.44) (P Next, we calculated the predictive value for diagnosis of MN of the most frequently mutated genes. In multivariable logistic regression, having two or more mutations (OR=4.07, P Among patients with a diagnosis of ICUS, 57 of 154 (37%) carried one or more mutations (Clonal Cytopenia of Undetermined Significance, CCUS). Patients with CCUS showed a significantly higher probability of developing MDS compared with those without evidence of clonality (HR=7.48, 10-year cumulative probabilities of progression: 96% vs 15% respectively, P Finally, the predictive values of mutation analysis were tested in the independent validation cohort of patients referred to our institution for suspected MDS, and were fully confirmed. In conclusion, selected mutated genes or co-mutation patterns may identify patients with high likelihood of having MDS or other MN. The definition of a category of CCUS allows to recognize with high sensitivity patients who do not fulfill diagnostic criteria but are at high risk of developing MDS. Taken together, these data suggest that mutation analysis on peripheral blood cells may significantly improve current diagnostic approach to patients with unexplained cytopenia. Disclosures Ogawa: Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding.

Background Current prognostic scoring systems in acute myeloid leukemia (AML) help predict outcomes in newly diagnosed patients at the time of diagnosis. In AML, achievement of complete remission (CR) is essential for the success of treatment. Therefore, development of a prognostic score for patients who enter first CR would be an important tool for physicians to guide their decisions for further treatment at the time of CR. Patients and Methods We developed a prognostic score based on data from 938 newly diagnosed adult AML (non-APL) patients (median age 59 years, range [18 - 88]) diagnosed and treated at the University of Texas MD Anderson Cancer Center, USA between [1999-2012], who achieved 1st CR/CRp and were not transplanted in 1st CR. Median follow-up was 22.3 months [range 1.4-165.1 months]. Information on MRC 2010 classification was available for 896 patients (156 (17.4%) patients in favorable, 550 (61.4%) in intermediate, and 190 (21.2%) in adverse group); ELN classification could be determined for 478 patients (160 (33.5%) in favorable, 88 (18.4%) in intermediate-1, 103 (21.5%) in intermediate-2, and 127 (26.6%) patients in adverse risk group. For survival analysis Kaplan-Meier method was used. Survival distributions were compared using log-rank tests. Univariable and multivariable Cox regression analyses were performed to evaluate an impact of clinically significant prognostic variables (P Results Based on multivariable analysis, age and MRC/ELN subgroup were statistically significant prognostic factors (p In "MRC +age" prognostic score four groups were identified: favorable, intermediate-1, intermediate-2 and poor (Table 1B). In "ELN+age" score five groups were identified: favorable, intermediate-1, intermediate-2, poor and very poor (Table 2B). According to "MRC+age" prognostic score, the 3- and 5-year overall survivals (OS), respectively were: 80% and 77% in the favorable group; 53% and 45% in the intermediate-1 group; 31% and 19% in the intermediate-2 group; and 8% and 6 % in the poor group. According to "ELN+age" prognostic score, the 3- and 5-year OS, respectively, were 78% and 77% in the favorable group; 68% and 50% in the intermediate-1 group; 46% and 37% in the intermediate-2 group; 27% and 22% in poor group; and 3% and 2% in the very poor group (Figure 1; A and B). Conclusion Despite the new discoveries in AML (DNA sequencing, genomic mutations, etc.) many clinics worldwide still don't have access to those tools and they stratify patients according to MRC or ELN classifications. The prognostic score presented above could be a powerful tool for physicians to risk-stratify patients with AML in first CR for post-remission therapy based on age and basic cytogenetic and/or molecular testing. Table 1. Prognostic score for AML in 1st complete remission according to Age and MRC classification Prognostic factor Coefficient Points Age Table 2. Prognostic score for AML in 1st complete remission according to Age and ELN classification MRC + age Total score Favorable 0 Intermediate-1 1-2 Intermediate-2 3 Poor 4-5 Table 3. Prognostic factor Coefficient Points Age Table 4. ELN + age Total score Favorable 0 Intermediate-1 0.5 Intermediate-2 2.5 Poor 3 Very poor 3.5 - 4.5 Figure 1. Cumulative rates of overall survival in patients with newly diagnosed AML in 1st complete remission according to A) MRC + Age prognostic score, and B) ELN + Age prognostic score Figure 1. Cumulative rates of overall survival in patients with newly diagnosed AML in 1st complete remission according to A) MRC + Age prognostic score, and B) ELN + Age prognostic score Disclosures Tamamyan: Conquer Cancer Foundation of the American Society of Clinical Oncology: Other: Long-term International Fellowship (LIFe). Cortes:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

2538 Background: NGR-hTNF, a selective antivascular agent, quickly and transiently modulates the release of systemic cytokines/chemokines. Intravenous infusion of NGR-hTNF is characterized by onset...

3038 Background: Antitumor effects of NGR-hTNF (N), a tumor-targeted antivascular agent, are driven at low dose by an early vessel stabilization that greatly enhances both intratumoral CT uptake and T-cell infiltration. Synergism with CT was shown in immunocompetent mice, but not in nude mice lacking functional T cells. Methods: By an individual patient pooled analysis of 396 patients (pts) from 7 ph II trials in 6 tumor types, we estimated the effects of baseline PBLC on the antitumor activity of N (with or without CT) and CT alone. Low dose N (0.8 μg/m2) was given in combination with CT in 171 pts. Control groups of 140 and 85 pts receiving N and CT alone, respectively, were also analyzed. CT was doxorubicin or a platinum-based regimen. In all trials, response to therapy was assessed every 6 weeks by RECIST. Endpoints of interest were response rate (RR, complete plus partial response), disease control rate (DCR, RR plus stable disease), duration of response (DOR) and progression-free survival (PFS). In logistic and Cox regression analyses, PBLC data were dichotomized in high or low levels by the median cutpoint (1.5/mL; 95% CI, 1.4-1.6). Multivariate models included age, sex, PS and tumor type as covariates. Results: In both N-alone and CT-alone groups, there was no statistically significant difference in treatment effect according to baseline PBLC. Conversely, high PBLC were related to better treatment outcome in the N plus CT group, compared to low PBLC. In this N plus CT group, high PBLC (vs low) were associated with higher RR (OR=2.8; 95% CI, 1.2-6.3; p=.01) and DCR (OR=2.6; 1.3-4.9; p=.004), and with longer DOR (HR=0.39; 0.16-0.96; p=.04) and PFS (HR=0.60; 0.43-0.85; p=.004). For high vs low PBLC, RR was 29% vs 14%, DCR 76% vs 57%, median DOR 8.2 vs 6.3 months, and median PFS 5.0 vs 3.0 months, respectively. On multivariate analyses, high PBLC remained an independent predictor of increased RR (OR=2.7; p=.01) and DCR (OR=2.6; p=.005), and improved DOR (HR=0.36; p=.04) and PFS (HR=0.60; p=.005). Conclusions: Consistently with preclinical data, these results highlight the potential value of PBLC in predicting tumor response to NGR-hTNF in combination with CT, which merits further clinical validation Clinical trial information: NCT00994097-NCT00484432-NCT00484276-NCT00484211-NCT00483509-NCT00483080-NCT01358071.

e13593 Background: NGR-hTNF, a selective antivascular agent, transiently modulates the systemic release of cytokines/chemokines. Intravenous infusion of NGR-hTNF is distinguished by an early on-target effect, IRR, which mostly consists of short-lived grade 1/2 chills. Methods: Incidence, predictors of development and relationships of IRR with treatment outcome were assessed in a pooled analysis of individual patient data from 5 phase II trials with NGR-hTNF. Global data set comprised 205 previously treated patients (pts) who had received NGR-hTNF 0.8 µg/m2 every 3 weeks (q3w) or weekly (q1w) alone in mesothelioma, colon and liver cancers or in combination with doxorubicin in small cell lung and ovarian cancers. In all trials, response to therapy (RECIST) was measured q6w until progression. Regression models estimated the effect size of IRR on response rate (RR; complete and partial response), disease control rate (DCR; RR plus stable disease) and progression free survival (PFS). Results: 137/205 pts (67%) experienced IRR on treatment, while 68 pts (33%) did not. In the IRR group, 63% of pts had grade 1 and 37% grade 2. By time to onset, 88% of pts developed IRR during first 6 weeks of therapy and 12% later. According to dosing schedule, IRR rates were higher with q1w than with q3w (85% vs 62%; p=.008). Baseline characteristics were (IRR vs non-IRR groups): median age: 66 vs 64; male: 50% vs 56%; PS ≥ 1: 34% vs 35%, range of prior lines: 1-4 vs 1-5. Among baseline characteristics, logistic regression analysis retained only a lower number of prior lines associated with higher IRR rates (OR=1.4; p=.02). Onset of IRR was related to better treatment outcome. RR was 12% in the IRR and 3% in the non-IRR group (OR=4.4; p=.05), while DCR was 50% in pts who had IRR and 34% in pts who did not (OR=2.0; p=.02). In pts with or without IRR, 6-month PFS was 21% vs 7% (HR=0.62; p=.001), respectively. Improvement in PFS time remained significant in a landmark analysis set at the 6-week time point (p=.004). On multivariate analyses adjusted for baseline variables, IRR independently predicted higher odds of tumor response (p=.05) and lower risk of progression or death (p=.009). Conclusions: Onset of IRR on NGR-hTNF treatment may predict subsequent clinical outcome. Clinical trial information: NCT00483080-NCT00483509-NCT00484211-NCT00484276-NCT00484432.