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Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) is an essential, highly conserved merozoite antigen that forms an important multi-protein complex (RH5/Ripr/CyRPA) necessary for erythrocyte invasion. CyRPA is a promising blood-stage vaccine target that has been shown to elicit potent strain-transcending parasite neutralizing antibodies. Recently, we demonstrated that naturally acquired immune anti-CyRPA antibodies are invasion-inhibitory and therefore a correlate of protection against malaria. Here, we describe a process for the large-scale production of tag-free CyRPA vaccine in E. coli and demonstrate its parasite neutralizing efficacy with commonly used adjuvants. CyRPA was purified from inclusion bodies using a one-step purification method with high purity (>90%). Biochemical and biophysical characterization showed that the purified tag-free CyRPA interacted with RH5, readily detected by a conformation-specific CyRPA monoclonal antibody and recognized by sera from malaria infected individuals thus indicating that the recombinant antigen was correctly folded and retained its native conformation. Tag-free CyRPA formulated with Freund’s adjuvant elicited highly potent parasite neutralizing antibodies achieving inhibition of >90% across diverse parasite strains. Importantly, we identified tag-free CyRPA/Alhydrogel formulation as most effective in inducing a highly immunogenic antibody response that exhibited efficacious, cross-strain in vitro parasite neutralization achieving ~80% at 10 mg/ml. Further, CyRPA/Alhydrogel vaccine induced anti-parasite cytokine response in mice. In summary, our study provides a simple, scalable, cost-effective process for the production of tag-free CyRPA that in combination with human-compatible adjuvant induces efficacious humoral and cell-mediated immune response.

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Plasmodium falciparum cysteine-rich protective antigen (CyRPA) is a conserved component of an essential erythrocyte invasion complex (RH5/Ripr/CyRPA) and a target of potent cross-strain parasite-neutralizing antibodies. While naturally acquired human RH5 antibodies have been functionally characterized, there are no similar reports on CyRPA. Thus, we analyzed the parasite-neutralizing activity of naturally acquired human CyRPA antibodies. In this regard, CyRPA human antibodies were measured and purified from malaria-infected plasma obtained from patients in central India and analyzed for their parasite neutralizing activity via in vitro growth inhibition assays (GIA). We report that, despite being susceptible to antibodies, CyRPA is a highly conserved antigen that does not appear to be under substantial immune selection pressure, as a very low acquisition rate for anti-CyRPA antibodies was reported in malaria-exposed Indians. We demonstrate for the first time that the small amounts of natural CyRPA antibodies exhibited functional parasite-neutralizing activity and that a CyRPA-based vaccine formulation induces highly potent antibodies in rabbits. Importantly, the vaccine-induced CyRPA antibodies exhibited a robust 50% inhibitory concentration (IC(50)) of 21.96 μg/ml, which is comparable to the IC(50) of antibodies against the leading blood-stage vaccine candidate, reticulocyte-binding-like homologous protein 5 (RH5). Our data support CyRPA as a unique vaccine target that is highly susceptible to immune attack but is highly conserved compared to other leading candidates such as MSP-1 and AMA-1, further substantiating its promise as a leading blood-stage vaccine candidate.

Immunotherapy has established itself as a promising tool for cancer treatment. There are many challenges that remain including lack of targets and some patients across various cancers who have not shown robust clinical response. One of the major problems that have hindered the progress in the field is the dearth of appropriate mouse models that can reliably recapitulate the complexity of human immune-microenvironment as well as the malignancy itself. Immunodeficient mice reconstituted with human immune cells offer a unique opportunity to comprehensively evaluate immunotherapeutic strategies. These immunosuppressed and genetically modified mice, with some overexpressing human growth factors, have improved human hematopoietic engraftment as well as created more functional immune cell development in primary and secondary lymphoid tissues in these mice. In addition, several new approaches to modify or to add human niche elements to further humanize these immunodeficient mice have allowed a more precise characterization of human hematopoiesis. These important refinements have opened the possibility to evaluate not only human immune responses to different tumor cells but also to investigate how malignant cells interact with their niche and most importantly to test immunotherapies in a more preclinically relevant setting, which can ultimately lead to better success of these drugs in clinical trials.

Abarrategi et al. summarize the methods used for human hematopoietic cell xenotransplantation and their milestones. They also discuss the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis., Xenotransplantation of patient-derived samples in mouse models has been instrumental in depicting the role of hematopoietic stem and progenitor cells in the establishment as well as progression of hematological malignancies. The foundations for this field of research have been based on the development of immunodeficient mouse models, which provide normal and malignant human hematopoietic cells with a supportive microenvironment. Immunosuppressed and genetically modified mice expressing human growth factors were key milestones in patient-derived xenograft (PDX) models, highlighting the importance of developing humanized microenvironments. The latest major improvement has been the use of human bone marrow (BM) niche–forming cells to generate human–mouse chimeric BM tissues in PDXs, which can shed light on the interactions between human stroma and hematopoietic cells. Here, we summarize the methods used for human hematopoietic cell xenotransplantation and their milestones and review the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis.

Myelodysplastic syndrome (MDS) is a hematological clonal stem cell disease. Recurrent splicing factors mutations are reported in 50% of MDS. Interestingly, mutations in the splicing factor gene SF3B1 are over-represented in MDS with ring sideroblasts (MDS-RS), co-occurring in up to 90% of patients. In MDS-RS, anemia is the major clinical manifestation. Erythropoiesis stimulating agents (ESAs) are used to treat anemia; however, the overall response rates are 20% to 40% with a duration of response of 18-24 months. New therapeutic options are needed to improve response to ESAs treatment and delay red blood cell transfusion, which are associated with acute myeloid leukemia progression and increase in morbidity. Mutations in SF3B1 modify the recognition pattern of the 3' splice site and lead to subsequent mis-splicing of its targets. To identify critical mis-splicing events involved in the erythroid differentiation blockage, we performed splicing analysis on RNA sequencing generated from hematopoietic stem/progenitor cells undergoing differentiation. Three MDS primary samples harboring SF3B1 mutations and three age-matched healthy donors cultured under normoxia and hypoxia conditions were initially used for the analysis. High depth RNA sequencing and differential splicing analyses using rMATS identified 2,845 mis-spliced events including 200 shared between hypoxia and normoxia conditions. Here, using a cohort of 42 MDS samples, we report the mis-splicing of the coenzyme A synthase (COASY) transcript. Heme synthesis relies on succinyl-CoA synthesis, and its production itself depends on the availability of cellular CoA. We thus hypothesised that COASY mis-splicing is a key driver of ineffective erythropoiesis in MDS-RS patients. In primary hematopoietic cells, COASY is upregulated during erythroid differentiation and its silencing in CD34 + cells severely impedes the generation of mature erythroid cells CD71 - CD235a + and causes disruption in heme production. Functional characterisations of the CRISPR-CAS9 edited K562 SF3B1K700E and the SF3B1-mutated HNT-34 cell lines confirmed that COASY mis-splicing impairs COASY protein synthesis that ultimately results in 60% loss of the protein. Metabolomic analysis showed that COASY mis-splicing depletes cells in CoA and succinyl-CoA metabolites, however this phenotype can be rescued by supplementation with vitamin B5, a CoA precursor. Consequently, we showed in vitro that saturating the 40% of remaining COASY enzyme with vitamin B5 or supplementing medium with its downstream by-product, succinyl-CoA, improved erythropoietic differentiation in MDS SF3B1mut patients. In summary, our results for the first time show that SF3B1 mutations induce coenzyme A synthase (COASY) transcript mis-splicing, that consequently leads to measurable defects in metabolites essential for heme biosynthesis. Our report reveals a novel critical role of COASY in regulating normal bone marrow erythropoiesis through control of succinyl-coA during human erythroid differentiation. Remarkably, partial loss of the coenzyme A synthase in MDS-RS patients leads to disruption in the erythroid lineage as well as heme deficiency, that can be rescued by exogenous treatment with vitamin B5 or succinyl-CoA. Therefore, vitamin B5 could represent a very attractive agent to combine with existing treatments in order to increase erythroid maturation and delay red blood cell transfusion dependency in MDS-RS patients. Graphical representation: SF3B1 mutant causes mis-splicing in COASY that results in loss of protein. Deficiency in COASY triggers a downregulation of succinyl-CoA that is involved in the rate limiting step of heme synthesis. Heme deficiency subsequently impairs erythroid differentiation. Treatment of MDS SF3B1 mutant cells with vitamin B5 (precursor of CoA), or succinyl-CoA, rescues erythroid differentiation. Figure 1 Figure 1. Disclosures Platzbecker: Geron: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; AbbVie: Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; StemLine: Consultancy; Takeda: Consultancy; Astex: Research Funding. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

The BM niche comprises a tightly controlled microenvironment formed by specific tissue and cells that regulates the behavior of hematopoietic stem cells (HSCs). Here, we have provided a 3D model that is tunable in different BM niche components and useful, both in vitro and in vivo, for studying the maintenance of normal and malignant hematopoiesis. Using scaffolds, we tested the capacity of different stromal cell types to support human HSCs. Scaffolds coated with human mesenchymal stromal cells (hMSCs) proved to be superior in terms of HSC engraftment and long-term maintenance when implanted in vivo. Moreover, we found that hMSC-coated scaffolds can be modulated to form humanized bone tissue, which was also able to support human HSC engraftment. Importantly, hMSC-coated humanized scaffolds were able to support the growth of leukemia patient cells in vivo, including the growth of samples that would not engraft the BM of immunodeficient mice. These results demonstrate that an s.c. implantation approach in a 3D carrier scaffold seeded with stromal cells is an effective in vivo niche model for studying human hematopoiesis. The various niche components of this model can be changed depending on the context to improve the engraftment of nonengrafting acute myeloid leukemia (AML) samples.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological clonal disorders. Here, we have tested the bone marrow (BM) cells from 38 MDS patients covering all risk groups in two immunodeficient mouse models: NSG and NSG-S. Our data show comparable level of engraftment in both models. The level of engraftment was patient specific with no correlation to any specific MDS risk group. Furthermore, the co-injection of mesenchymal stromal cells (MSCs) did not improve the level of engraftment. Finally, we have developed an in vitro two-dimensional co-culture system as an alternative tool to in vivo. Using our in vitro system, we have been able to co-culture CD34+ cells from MDS patient BM on auto- and/or allogeneic MSCs over 4 weeks with a fold expansion of up to 600 times. More importantly, these expanded cells conserved their MDS clonal architecture as well as genomic integrity.

Evidence of the presence of abnormalities in the primitive hematopoietic stem cells, as well as functional analysis, has demonstrated that Myelodysplastic syndrome (MDS) is propagated by rare and distinct human MDS propagating cells (MDS-PCs) in 5q- and in refractory anemia with ring sideroblastic (

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T-cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in-vitro expandability for potential clinical use. Using mass cytometry (CyTOF) and an unbiased multidimensional analytical approach, we identified two specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene-expression, expandability and function. Treg subpopulation B, predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4 and CD45RO within FOXP3hi, CD127lo Tregs), expresses the IL- 2/STAT5 pathway and cell-cycle commitment genes. Furthermore, in-vitro expanded Tregs become functional and with the characteristics of Treg subpopulation B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2 sensitive and expandable in-vitro, suggesting novel therapeutic approaches such as low dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

The role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using “next-generation” sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chroni myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. “Deeper” sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34+ and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Despite the recent evidence of the existence of myelodysplastic syndrome (MDS) stem cells in 5q-MDS patients, it is unclear whether haematopoietic stem cells (HSCs) could also be the initiating cells in other MDS subgroups. Here we demonstrate that SF3B1 mutation(s) in our cohort of MDS patients with ring sideroblasts can arise from CD34+CD38−CD45RA−CD90+CD49f+ HSCs and is an initiating event in disease pathogenesis. Xenotransplantation of SF3B1 mutant HSCs leads to persistent long-term engraftment restricted to myeloid lineage. Moreover, genetically diverse evolving subclones of mutant SF3B1 exist in mice, indicating a branching multi-clonal as well as ancestral evolutionary paradigm. Subclonal evolution in mice is also seen in the clinical evolution in patients. Sequential sample analysis shows clonal evolution and selection of the malignant driving clone leading to AML transformation. In conclusion, our data show SF3B1 mutations can propagate from HSCs to myeloid progeny, therefore providing a therapeutic target., Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders with diverse phenotypes and can derive from hematopietic stem cells after the acquisition of specific somatic aberrations. In this study, the authors show that MDS initiating cells in some cases of sideroblastic anemia with SF3B1 mutations, can arise from hematopoietic stem cells.

Summary Background A mechanism for clonal growth advantage in isolated del(5q) disease remains elusive. CSNK1A1 resides on the critically deleted region, and deletion of this gene has been shown in mouse knockout and transplantation studies to produce some characteristics of bone marrow failure, including a proliferative advantage. We aimed to establish the frequency, nature, and clinical association of CSNK1A1 mutations in patients with myelodysplastic syndrome and associated myeloid neoplasms. Methods Between June 1, 2004, and May 31, 2014, in King's College (London, UK), we did whole-exome sequencing of five patients with isolated del(5q) followed by targeted screening for CSNK1A1 mutations and 20 myelodysplastic syndrome-associated mutations in 245 additional patients with myeloid neoplasms. All patients met present WHO diagnostic criteria for myelodysplastic syndrome and other related myeloid neoplasms. Findings 39 (16%) of 250 patients with myeloid neoplasms had isolated del(5q), of whom seven (18%) had CSNK1A1 mutations. All these mutations were missense and presented in a highly conserved region that is implicated in ATP catalysis. Serial sampling and response to lenalidomide treatment showed that CSNK1A1 mutations were highly associated with the del(5q) clone. Only one patient with a CSNK1A1 mutation showed complete cytogenetic response to lenalidomide. Four (57%) of the seven patients carrying a CSNK1A1 mutation showed disease progression coupled with an increase in mutant allele burden (all four were on lenalidomide). We detected coexisting myelodysplastic syndrome-related gene mutations in patients with CSNK1A1 mutations, including TP53 . Interpretation Similar to the effect of TP53 mutations on progression of del(5q) abnormality, mutant CSNK1A1 also gives rise to a poor prognosis in del(5q) abnormality, for which a coupled increase in P53 activation is suggested. CSNK1A1 mutations in del(5q) disease are important in the context of therapeutic manipulation and need incorporation into future prospective studies. Funding Leukaemia and Lymphoma Research.

The distinction between acquired aplastic anemia (AA) and hypocellular myelodysplastic syndrome (hMDS) is often difficult, especially nonsevere AA. We postulated that somatic mutations are present in a subset of AA, and predict malignant transformation. From our database, we identified 150 AA patients with no morphological evidence of MDS, who had stored bone marrow (BM) and constitutional DNA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. The initial cohort of 57 patients was screened for 835 known genes associated with BMF and myeloid cancer; a second cohort of 93 patients was screened for mutations in ASXL1, DNMT3A, BCOR, TET2, and MPL. Somatic mutations were detected in 19% of AA, and included ASXL1 (n = 12), DNMT3A (n = 8) and BCOR (n = 6). Patients with somatic mutations had a longer disease duration (37 vs 8 months, P.04), and shorter telomere lengths (median length, 0.9 vs 1.1, P.001), compared with patients without mutations. Somatic mutations in AA patients with a disease duration of6 months were associated with a 40% risk of transformation to MDS (P.0002). Nearly one-fifth of AA patients harbor mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS.

Background The presence of diverse spectrum of mutations in myelodysplastic syndromes (MDS) has largely revolutionized the understanding of the disease. Although certain mutations have doubtful prognostic significance, the adverse impact of TP53 mutation is irrefutable in all publications. We and others have shown the strong correlation between presence of TP53 mutation and overexpression of p53 by immunohistochemistry (IHC). This evidence provides the rationale for the evaluation of p53 overexpression which is available in routine diagnostic workup, as a surrogate marker to predict TP53 mutations. Additionally, we predicted that the overexpression of p53 might be an independent prognosticator, even in the absence of TP53 mutations or RPS14 haploinsufficiency, in low risk MDS. In our study,we analyzed the p53 protein expression in a cohort of 277 'lower risk' MDS patients and compared the clinico-pathological features, overall survival and risk of progression and evolution to AML with respect to p53 IHC status. Methods This study enrolled 277 adult low risk MDS patients seen at King's College Hospital between March 2009 and February 2016. All marrow samples were assessed for p53 expression (intensity and proportion of cells) and scored using a Modified Quick Score (MQS). MQS ≥2 was chosen to define p53-positive staining. The median follow-up was 18.7 months [95% confidence interval (CI), 16.5-21.0 months]. Results According to WHO categories, the predominant group was RCMD (78%). Patients were either IPSS low (n=134, 48%) or int-1 (n=91, 33%). We included 52 (19%) pts for whom the IPSS category was not available, due to the lack or failed cytogenetic data, but these were low-risk WHO categories with no excess of blasts. IPSS-R categories were very low 103 (37%), low 77 (28%), intermediate 38 (14%) and high risk 7 (3%). Overall, 19 (7%) patients progressed to AML and 41 (15%) patients died of which 25 (61%) were disease-related deaths. Of the 277 patients, 148 (53%) showed p53 protein expression with MQS ≥ 2 and were considered "p53-positive" patients (p53+). The p53 stain intensity was negative in 125 (45%), weak in 84 (30%), moderate in 40 (14%) and strong in 28 (11%). Seventy six (28%) patients had >5% p53 staining cells. p53 expression correlated with a higher age at diagnosis (median age 64 vs. 60 years, p=0.01). lower haemoglobin levels (9.8 vs 11 g/dL, p=0.002), but a higher platelets count (139 vs 99 x109/L, p =.003) (Table 1). This significance persisted even on exclusion of patients with 5q- and MDS/MPN. P53+ did not correlate with any cytogenetic risk group or degree of fibrosis. Sequencing data was available in 121 patients with 63 harbouring somatic mutations. Among these patients, only 3 showed the TP53 gene mutation; all of them were classified as positive when assessed for p53 stain (MQS≥2), showing that the nuclear staining reflects underlying TP53 mutations in such cases. Additionally, presence of SF3B1 mutation correlated strongly with p53+ (p=0.02). Patients with p53+ had a significantly shorter OS compared with those with p53- (figure 1a) with 2yr and 4yr survival probability of 87% &68% vs 95% & 90% , respectively (p=0.03 ). Analysis of progression-free survival (progression to AML, cytogenetic evolution, increase of blasts or fibrosis) showed that patients with p53 expression had a significantly shorter PFS compared to patients without p53 expression (Figure 1b): 2yr & 4yr PFS 70% & 54% vs 85% &75% respectively, p=0.01). Multivariate analysis confirmed the independent prognostic value of p53 expression for PFS (p=0.013, HR 2.6, 95% CI 1.2-5.2). Conclusion In conclusion, our study showed that p53 overexpression by IHC, represented as MQS cutoff ≥ 2, correlates with features of poor prognosis, such as more advanced age at diagnosis, lower haemoglobin levels and shorter overall survival, adversely impacting progression-free survival. p53 IHC in MDS patients may represent an important, easily available, cheap and applicable prognostic tool and it should be considered for auxiliary analysis when determining the therapeutic options for a patient. The p53+ predicts poor outcome, especially disease progression, independent of the TP53 mutation status, indicating an alternate yet undefined pathway for p53 overexpression. Figure 1(a) Disease-related OS (a) and PFS (b) in patients with low-risk MDS according to their p53 expression-status Figure 1(a). Disease-related OS (a) and PFS (b) in patients with low-risk MDS according to their p53 expression-status Figure 1(b) Figure 1(b). Disclosures No relevant conflicts of interest to declare.

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

INTRODUCTION The cohesin complex is a multimeric protein complex reported to have putative roles in post-replicative DNA repair, sister chromatids cohesion and transcriptional regulation. Somatic mutations in the genes that constitute the cohesin complex (NIPBL, SMC1A, SMC3, and HDAC8) give rise to the debilitating disease, Cornelia de Lange Syndrome. The cohesin genes are mutated in several cancers, the highest incidence reported in bladder cancer followed by myeloid cancers such as myelodysplastic syndrome (MDS). Inhibition of PARP (Poly ADP Ribose Polymerase) activity has been shown to selectively target AML patients with DNA repair defects (Gaymes et al 2009). Discovery of mutations that confer sensitivity to PARP inhibitors have the potential to identify candidates for therapeutic intervention. Mutation in genes involved in epigenetic regulation and RNA splicing account for 70% of the mutations reported in MDS. Mutations in cohesion complex have been reported, but have not been systematically studied in a large and uniformly treated patient cohort. To characterise still further the mutations that shape the MDS phenotype, 154 MDS patients that had been screened for myeloid gene mutations using the 22 gene panel (Syed et al 2013) were re-screened for mutations in four of the cohesin complex genes (SMC1A, SMC3, RAD21 and STAG2). The functional consequences of defective cohesin were also studied using shRNA silencing. METHODOLOGY Nextera sequencing was used to sequence all the coding exons of SMC1A, SMC3 and RAD21 genes, whilst STAG2 was sequenced using the Roche 454 platform. To further elucidate the role of cohesin complex in MDS, lentiviral shRNA silencing of STAG2 was achieved in the AML cell line, U937 and effects of knockdown was subsequently determined through functional assays. RESULTS Sequencing of the cohesin complex genes revealed mutations in 6% of MDS patients of which 4% had STAG2, 1% had SMC3 and0.6% patients had SMC1A mutations. None of the patients studied had a RAD21 mutation. All detected mutations were confirmed by independent PCR and Sanger Sequencing. For STAG2 mutations, a constitutional source of DNA was also used to confirm the mutations, (skin biopsy (n=2), T cells (n=3). 5/6of the STAG2 mutationswere nonsense with an average allelic burden of 33% indicative of a heterozygous mutation. Mutations in SMC3 were reported in two patients; a frameshift mutation found in the coiled coiled domain and a substitution in the 3' splice acceptor site. The average allelic burden for both the mutations was 20%. A missense mutation was identified in SMC1A in one patient with an allelic burden of 13%. (3/9) patients mutations in STAG2 gene coexisted with SRSF2 and ASXL1 mutations. The patients with cohesion mutations had a significantly lower progression free survival as compared to patients without mutation (Figure 1A) Lentiviral shRNA silencing of 75% and 66% for STAG2 was achieved in U937 cells using two shRNAs, confirmed both by q-PCR and Western blot. shRNA silencing of STAG2 also resulted in reduced expression of partner cohesin sub-unit, RAD21 confirmed by western blot. Strikingly, treatment of shRNA silenced U937 with PARP (Poly-ADP-Ribose Polymerase) inhibitors resulted in 28% cytotoxic reduction compared to scrambled control (Figure 1B). Furthermore, cytotoxicity was independent of Homologous Recombination DNA repair competency as recruitment of RAD51 to damaged sites was not altered in shRNA silenced STAG2 U937 cells upon challenge with PARP inhibitors. CONCLUSION In this study, cohesin complex mutations were found in 6% of MDS patients with STAG2 being the most prevalent. We also report the co-existence of cohesin mutations with mutations in ASXL1 and SRSF2. The cohesin mutant allelic burden in these patients was lower compared to ASXL1 and SRSF2 mutations suggesting that cohesin mutations co-exist as passenger mutations alongside mutations that drive clonal evolution like ASXL1 as reported previously (Thota et al 2014). The observation that loss of STAG2 confers PARP inhibitor sensitivity is of major clinical importance. Identification of a cohort of MDS/AML patients with cohesin mutations would signify a major development in the identification of candidates for PARP inhibitor therapy. Disclosures Kulasekararaj: Alexion: Consultancy.

Autoimmune disorders (AIDs) are commonly observed in up to 30% of myelodysplastic syndromes (MDS) patients. Clinical manifestations such as acute neutrophilic febrile dermatosis, rheumatoid arthritis, inflammatory bowel disease, pulmonary infiltrates and peripheral polyneuropathy, precede or accompany the diagnosis of MDS. In fact, large-scale epidemiologic studies have suggested that patients with AIDs have an elevated risk of developing MDS. To gain more insight into the association of MDS and AIDs, bone marrow mononuclear cells available from 202 patients were sequenced for 'inflammsome' pathway genes NLRP3 and MYD88, which have been shown to carry mutations in various AIDs. According to WHO classification, the patient cohort included 49% RA/RARS/RCMD, 24% RAEB, 6% 5q syndrome, 10% AML, 6% MPD/MDS and 5% tMDS. Median age of the cohort was 62 years (IQR 54.8-62.3). Autoimmune data was available on 129 patients and 49/129 patients had evidence of AID. 31 patients were diagnosed with MDS-associated 'Sweets Syndrome' AID. Myeloid neoplasm-related 22 gene panel mutation data was available for 171 patients. Initially all protein coding exons of NLRP3 and MYD88 genes were sequenced in 31 cases diagnosed with MDS-Sweets syndrome. Targeted sequencing (Miseq, Illumina) revealed NLRP3 variants in 6/31 cases. NLRP3 heterozygous variants detected were V200M (n=1/31, 3%), Q705K (n=4/31, 13%) and T954M (n=1/31, 3%). Two of these variants (V200M and Q705K) have been previously described as pathogenic and linked with various autoimmune disorders1,2. Variant T954M is reported in COSMIC v73 database as a confirmed somatic mutation. The frequency of these variants in general European population (NHLBI ESP 2015) varies in-between 0.6% to 5% (V200, rs121908147 MAF= 0.7%; Q705, rs35829419 MAF= 5% and T954, MAF= NLRP3 heterozygous variants V200M and Q705K were observed in 2.3% (n=4/171) and 12% (n= 20/171), of the patients screened, respectively. T954M variant was not detected in the follow-up cohort. Sequencing of the paired constitutional DNA (CD3+ lymphocytes/ Skin biopsy), where available (n=10), confirmed that V200M and Q705K are inherited SNPs. The frequency of these NLRP3 variants observed among our study population is marginally higher than previously described in the general European population (Pearson chi-square test, P >0.05) In order to detect the effects of the NLRP3 variant on the inflammasome pathway, we quantified the serum cytokine (IL1β, IL18, TNFα and INFγ) levels using ProcartaPlex Human Cytokine & Chemokine Panel 1A (34 plex, eBioscience) kit in patients with NLRP3 variants (MDS/sweets syndrome, n =3) vs NLRP3 wildtype (MDS-Sweets syndrome, n=5; MDS/non-sweets syndrome, n=2). No difference was observed in the serum cytokine levels between two patient groups. Next, we investigated the mRNA levels of the NLRP3 and other genes that lie downstream of the NLRP3 inflammasome pathway (IL1β, IL18, TNFα, CASPASE-1 and INGγ). 34 patients (NLRP3 variant, n= 22 and NLRP3 Wildtype, n= 12) were included in this analysis. An increased trend in the IL18 mRNA levels was observed in patients with NLRP3 variant vs NLRP3 wildtype. In conclusion, our study shows that the NLRP3 variants are enriched in MDS patients with AIDs compared to historical data on the general population. Patients with NLRP3 variants have an increased levels of IL18 mRNA levels. A larger study to explore the inflammasome pathway is needed to define the molecular pathogenesis of the autoimmunity associated with MDS. Identifying the casual genomic variants of AIDs in MDS may provide relevance to development of novel therapeutic strategies. References 1. Verma D, Sarndahl E, Andersson H, et al. The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1beta and IL-18 production. PLoS One. 2012;7(4):e34977. 2 . Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat Genet. 2001;29(3):301-305. Disclosures Kulasekararaj: Alexion: Consultancy. List:Celgene Corporation: Honoraria, Research Funding.

Background Shortened telomeres are seen in approximately a third of patients with idiopathic aplastic anemia (AA) and they also define risk of relapse, clonal evolution and overall survival. Constitutional pathogenic mutations in the telomere gene complex (TGC) are associated with very short telomeres (typically Methods We report results using telomere length (TL) measurement by quantitative-real time PCR (qPCR) as a screening tool to identify patients for further mutation analysis on a customised panel of 10 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1 and WRAP) using deeply parallel sequencing in a cohort of patients with AA. Using a Polyphen score that predicts a variant to be possibly pathogenic, we have used telomere repeat amplification protocol (TRAP) assay to increase the robustness of classifying a variant as more likely pathogenic, where family history was unrevealing. TRAP assay was performed by introducing the variants into W138V13 cell line and comparing telomerase expression in them to wild type TERT and TERC expression of telomerase. Results From the King's College Hospital database, we screened 295 patients with AA for TL using qPCR. The median age of the cohort was 44.2 years (range18.2- 83.4) with male/female ratio of 57:43. 189 patients (64%) had TL < 10th centile and 111 (37.6%) had TL We identified a further 9 novel variants which were predicted to be possibly pathogenic, but have not been reported pathogenic in literature, as yet. All 9 patients with the novel variants had TL Conclusion Two thirds of patients with AA have TL Figure 1. Telomere length expressed as T/S ratio against age for the different cohorts Figure 1. Telomere length expressed as T/S ratio against age for the different cohorts Figure 2. Expression of telomerase activity in novel TERT and TERC variants compared to Wild-type (positive control) and Mock (negative control) Figure 2. Expression of telomerase activity in novel TERT and TERC variants compared to Wild-type (positive control) and Mock (negative control) Disclosures Kulasekararaj: Alexion: Consultancy.

Shortened telomeres are seen in approximately a quarter of patients with idiopathic pulmonary fibrosis (IPF) and aplastic anemia (AA) and may have a role in disease pathogenesis. Mutations in the telomere gene complex (TGC) are less frequently seen in IPF (1%) and 5-10% in AA. Novel mutations affecting telomere function are increasingly being reported and involve the telomerase genes, the shelterin and helicase complexes, but their prognostic significance remains undefined. We report results on telomere length (TL) using multiplex quantitative real-time PCR (qPCR) and mutation analysis on a customised panel of 10 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1 and WRAP) using deeply parallel sequencing in a cohort of 69 patients with AA and IPF and their effects on clinical variables and response rates. 69 patients (49 with AA, 14 with IPF and 6 with a bone marrow failure syndrome (BMFS) and pulmonary fibrosis; M/F =43/26) with a median age of 49.7 years (range 9-81) had TL (T/S ratio) tested using qPCR on peripheral blood mononuclear cells at diagnosis or pre-treatment. Massively parallel targeted sequencing of the TGC was performed in all cases and constitutional mutations identified using the exome variant server. Clinical variables studied included hemoglobin (Hb) level, neutrophil count (ANC), platelet count (PC), LDH and albumin levels, and response rates to immunosuppressive treatment (IST). In patients with AA and other BMFS, red cell and platelet transfusion dependency, bone marrow cellularity (1) on bone marrow (BM) staining were correlated with TL and presence of any TGC mutations. In patients with IPF, corrected transfer factor (Kco) was correlated with TL and mutation analysis. 44/69 (64%) had TL below the 1st centile and 20 (29%) of them had TL very significantly below the 1st centile compared to a normal cohort (n=180) of age-matched individuals. Median Hb was (11g/dl), ANC was 1.69 and PC was 96x109/l. There was no statistical difference between patients whose TL was below or above the 1st centile for Hb levels, reticulocyte count or PC. 24 patients (34.7%) were red cell transfusion dependent and 15(21.7%) needed platelet transfusions. 42/55 (76.3%) had BM cellularity < 30% and was not statistically significant between patients with TL above or below the 1st centile. Similarly there was no statistical difference in LDH and albumin levels between the two groups. Equal number of patients were treated with IST between the two groups and response rates were similar. Median levels of Kco (0.95) were identical in IPF patients. 62 (89.8%) of patients were alive at a median follow up of 2.15 years (range 0.3-26.8). TGC mutations were seen 16 patients (23%) in the entire cohort with 13/44 (30%) were seen where TL was below the 1st centile as compared to 3/25 (12%) where TL was above the 1st centile (p In our cohort, about two-thirds of patients had TL below the 1st centile. TL, however, does not affect blood counts, BM cellularity and p53 staining, red cell or platelet transfusion dependency or Kco in patients with IPF. A higher proportion of patients with TL below the 1st centile were found to have TGC mutations, although statistical significance was not achieved. Missense mutations in TERT were the most frequent followed by mutations in TERC. Patients with TL Table Variable TL < 1st centile TL > 1st centile Male/Female (Total) 29/15 (44) 14/11 (25) Age < 40 years at diagnosis 23 (52.2%) 7 (28%) TGC mutations % 30 12 Hb (g/dl) 11 11.2 Reticulocyte count ( x 10*9/L) 52 57 ANC ( x 10*9/L) 1.69 2.79 PC ( x 10*9/L) 90 139 Albumin g/dl 42 42 LDH (IU) 211 210 AA/BMFS 37 18 Red cell transfusion dependency % 36 44.4 Platelet transfusion dependency % 18.1 38.8% Cytogenetic abnormality % 15.6 0 BM cellularity < 30% % 77.7 72.2 P53 (Modified quick score >1) % 34.7 40 Treatment with IST Response % 23 (62.1%) 69.5% 11 (64.7%) 72.7% IPF 7 7 Kco 0.95 (0.6-1.9) 0.95 (0.8-1.2) Disclosures Kulasekararaj: Alexion Pharmaceuticals: Speakers Bureau.

Introduction 5-azacitdine (aza) treatment in myelodysplastic syndrome (MDS) induces a response rate of 40-45% and prognostic factors for response and survival remain still largely unknown. Presence of mutant TET2 has been shown to predict better response to aza and the recently described French Azacitidine prognostic score segregates patients into 3 groups with varying median overall survival. Although a plethora of somatic mutations have been described in MDS, none has been consistently shown to be prognostically important in the context of response to hypomethylating agent(s). Patients and methods To identify the mutation signature associated with aza response, we undertook screening of 24 myeloid genes (splicing, epigenetic, transcription factors, STAG2, TP53) in 66 MDS patients treated with aza at our institution over a period of 2004-2012. Mutation analysis was done by deep (454 FLX) and Sanger sequencing. SNP-6 karyotyping was also performed in a subset to correlate with mutation status. Responses were assessed as per the international working group for MDS criteria. The median age was 67 years (range 36–87 years), median number of courses 7(range 2–42), 79% of patients belonged to int-2/high risk IPSS category. WHO category subtypes were; RA/RARS-2; RCMD-9; RAEB-39; s-AML (evolved from pre-existing MDS) -5, therapy related myeloid neoplasm (t-MDS/t-AML) -8 and CMML-3.IPSS cytogenetic subgroups were, good risk: 22, intermediate: 7, and poor risk: 37. One fourth of patients had received prior therapy, with only two receiving low dose cytarabine. Median time from diagnosis to aza treatment was 8.6 months. The overall response rate (ORR) to Aza was 47% (31/66) with complete response (CR) 17%, partial response (PR) 11%, marrow CR (mCR) 12% and stable disease with hematological improvement (SD-HI) 7%. Results Candidate mutations were seen in 82% (54/66) of patients, with the more than half harboring ≥2 mutations each. The most frequently (≥5 %) mutated genes being ASXL1 (29%), TP53 (23%), TET2 (14%), DNMT3A (12%), SRSF2 (12%), EZH2 (11%), NRAS (8%), U2AF1 (8%), IDH2 (8%), RUNX1 (8%), CCBL (6%) and FLT3-ITD (5%). On univariate analysis presence of EZH2 mutations predicted for a better ORR compared to wild type EZH2 (21% vs. 3%, p Patients with any mutation had a worse outcome compared to patients with no mutations (p=0.03, med OS 12.4 vs. 20.2 months). TP53 mutations were detected and quantified by high throughput sequencing in poor risk IPSS cytogenetic group (15/37) and were absent in patients with normal cytogenetics and isolated chromosome 7 abnormalities. Sequential samples were available in 9 patients and changes in clone sizes were correlative of treatment response, with 3 patients achieving cytogenetic response with concurrent reduction in clone size but the responses were transient. The TP53 mutation status correlated strongly with finding aberration of chromosome 17p by SNP array and positive p53 antibody staining on bone marrow trephine. On univariate analysis, TP53 mutations had an adverse impact on OS (P=0.0001, med OS 9.7 vs. 20.2 months). Age, IPSS cytogenetic risk group, WHO subtypes, overall response rate (ORR),progression to AML, TP53 mutation status and presence of EZH2 ,ASXLI,TET2,DNMT3A, SRSF2,NRAS mutations were used as co-variables in multivariate analysis.TP53 mutation status (HR=7.9(3.9-17.8), p=0.0001) and DNMT3A mutations (HR=2.5 (1.03-6.2), p=0.04) were established as independent prognostic variables influencing OS. Additionally, ORR to aza treatment imparts a better survival advantage (HR=0.3 (0.16-0.57),p=0.0001) Conclusion This study shows that gene mutations are frequent in all cytogenetic subgroups of MDS. TET2 mutations did not predict response to aza contrary to published reports, which could be due to differences especially as low dose cytarabine was infrequently used in our cohort. TP53 mutations do not correlate with response rate but imparts a dismal prognosis. In conclusion, although we have screened comprehensively we do not identify a mutational profile predictive of response to azacitdine. Disclosures: No relevant conflicts of interest to declare.

Introduction Defects in pre-mRNA splicing have recently been implicated in the pathogenesis of myelodysplastic syndrome, particularly RARS where SF3B1, a key component of the spliceosome machinery is mutated in up to 83% of the cases. Moreover, as homozygosity is the exception to the rule for these mutations and SF3B1 homozygous systems have been previously reported as lethal, then such aberrations are most certainly not benign bystanders and are likely to play a significant role in pathogenesis of the disease. Besides the strong correlation of SF3B1 mutations and the presence of ringed sideroblasts, the mechanism through which these mutations affect their target cells, distort the RNA splicing process and subsequently lead to disease still remains unclear. Methodology In order to gain insight into the effects of SF3B1 mutations on the engraftment kinetics of haemopoietic stem cells, bone marrow CD34+ cells from RARS patients (n=4) with SF3B1 mutations (with mutant allele burden 20% to 48%) were transplanted in NOD/SCID/interleukin-2 receptor γ chain–null (NOD/SCID/IL2rγ−/−) mice (1-3 mice/patient), and were also plated in committed progenitor (colony-forming cell, or CFC) and primitive progenitor (long-term bone marrow culture-initiating cell, or LTC-IC) cultures. Following on, whole-exome sequencing (Illumina) was performed on pre-transplant (bone marrow CD34+/TNC), post-transplant week-24 (human CD45+CD33+) and LTC-IC week-5 samples. In addition, we also performed whole-exome sequencing on FACS-purified HSCs (Lin- CD45+CD34+CD38- CD45RA-CD90+CD49f+), MPPs (Lin- CD45+CD34+CD38-CD45RA-CD90-CD49f-), GMPs (Lin- CD45+CD34+CD38+CD135+ CD45RA+) and MEPs (Lin- CD45+CD34+CD38+CD135-CD45RA-) from 3 of these patients. Results Our data demonstrates that the SF3B1 mutations arise from the early HSCs and are propagated without diminution in the mutant allele burden from HSC to more committed myeloid progenitors (i.e. MPPs, GMPs and MEPs). We also show, that the transplantation of bone marrow CD34+ cells derived from SF3B1 mutant RARS patients into mice leads to persistent long-term engraftment (0.01% - 4%, up to 24 weeks) of only myeloid cells but not lymphoid cells, irrespective of the type of SF3B1 mutation. The RARS-originated cells recovered from the human cell-engrafted mice maintained its genotypic characteristics, as demonstrated by the presence of SF3B1 mutations which remained largely unchanged between the pre- and post-transplant samples. This phenomenon was also observed in LTC-IC and CFC culture experiments where the SF3B1 mutant allele burden was maintained at the same level. In all analysed samples, there was no difference in the SF3B1 mutation burden between the LTC-IC with cytokines or without cytokines, and CFC under normoxic or hypoxic conditions. Whole-exome sequencing also revealed additional 20-60 known and/or novel candidate somatic mutations which were present concurrently in mice, LTC-IC and/or CD34+ (pre-transplant) cells for each RARS experiment. These include genes involved in epigenetic modifications, cell signalling/transcription regulation, DNA maintenance and cytoskeletal organization. These candidate mutations were either maintained at the same mutant allele burden or reduced/increased between the pre-transplant, post-transplant and LTC-IC, and are likely to be the sub-clones of SF3B1 clone. For example, in one RARS case JAK2 V617F mutation changed from 20% at pre-transplant to 9% (average mutant allele burden in mice, n=3) at post-transplant and 10% in LTC-IC. However, DNMT3A W581S mutation present in another RARS case was largely maintained i.e. 44% at pre-transplant, 38% at post-transplant (n=1) and 43% in LTC-IC. Conclusion These results demonstrate that SF3B1 mutations arise from the early HSCs, persist in the MEPs/GMPs, and retain their engraftment capacity in-vivo as well as their long-term proliferation potential in-vitro. Our murine xenograft RARS model comprised of human myeloid progenitor and mature cells, the majority of these cells, if not all, originated from the patients original clonal HSC pool as exhibited by its aberrant genotypic characteristics. Furthermore, this study also suggests that the cells carrying the SF3B1 mutations have a clonal growth advantage as compared to normal cells present in these RARS patients, and is therefore likely to contribute to the RARS disease phenotype. Disclosures: Bonnet: Janssen and Janssen: Collaborative Project and Consultant Other; Genetech: Collaborative project, Collaborative project Other.

Abstract 3826 Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders characterized by ineffective haematopoiesis. Age, gender, mutagen exposure and telomere length have been linked to MDS. Previous studies have demonstrated that patients with MDS have shortened telomeres compared to healthy controls, based on measurements obtained from either peripheral blood mononuclear cells, peripheral blood granulocytes, bone marrow, and/or CD34+ stem cells, indicating that individuals with shorter telomeres may be at increased risk of developing MDS. To investigate the association between telomere length and pathogenesis of MDS, we measured the telomere length (T/S ratio) by a multiplex quantitative real-time PCR in bone marrow mononuclear cells of 307 MDS patients and PBMCs of 182 healthy controls. In the assay, the relative telomere length is measured by the fluorescence signal of telomere amplification normalized to the signal obtained from a single copy gene, albumin. The median age of patients was 64 years (range 17–89 years). The median haemoglobin levels was 9.9 g/dl(IQR 8.6–11.6), neutrophil 1.4 × 109 /l (IQR 0.6–2.99) and platelet 98x 109 /l (IQR 39–187).The WHO subtypes were RA/RARS/Isolated de5q; 50 (16%), RCMD/RCMD-RS; 116 (38%), RAEB 1/2; 70 (23%), AML secondary to MDS; 29(9%),therapy related MDS; 18 (6%) and MDS/MPD; 24 (8%). IPSS cytogenetic risk groups were; good risk-199(65%), intermediate -34 (11%) and poor risk-55(18%) and cytogenetics failed in 19 patients (6%). The IPSS categories were, low risk: 80(26%), intermediate-1:97(32%), intermediate-2:50 (16%), high risk: 26 (9%) and 54(18%) patients were not evaluable (proliferative CMML and MPD/MDS).Transfusion dependency was present in 141(46%) patients. Progression to AML occurred in 68 patients (20%). In healthy controls (n=182; age range: 2–90 years), the T/S ratios measured in PBMCs demonstrated a progressive decline with ageing (Y=2.3–0.014X; R2=0.2417; P < 0.0001). The median telomere length was 0.97(range 0.3–2.8).In patients with MDS, T/S values did not show a correlation with age (P=0.327). Neither statistically difference in T/S values was observed between male and female patients (P=0.976). However, compared to PBMCs of the age-matched healthy controls, the mean T/S value obtained from BMNC of the MDS patients was significantly lower (P MDS patients with complex cytogenetics(n=35, med T/S value 0.919) had a shorter telomere length compared to patients with normal cytogenetics (n=204, med T/S value 1.02).We did not observe any statistically significant difference in T/S values in patients with isolated 5q, trisomy 8, monosomy 7. We could not find any association between short telomere and MDS patients with TP53 mutation. In multivariate logistic regression, telomere length was associated with transfusion dependency (P1.2, though this was not statistically significant. This study shows that MDS patients have short telomeres compared to age matched controls and the relative telomere length is strongly associated with IPSS risk group and transfusion dependency. Telomere attrition seen in patients with MDS could lead to destabilization of genome and subsequent chromosomal instability. This study shows shortening of telomeres associated with complex cytogenetics, transfusion dependency and also in patients with intermediate-2/high risk MDS. Disclosures: Elebute: Alexion: Honoraria. Mufti:Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 748 NCL, AA, AK & AS contributed equally to the study A large number of acquired mutations have been identified in haematological malignancies in recent years. An increasing number of targets present a new problem for diagnostic molecular pathology laboratories. It is not practical for a laboratory to design increasing numbers of gene specific assays for these targets. For these reasons a single assay capable of detecting a large number of gene mutations is desirable. Here we describe a next generation sequencing approach using the Roche 454 platform which is capable of the simultaneous mutation analysis of 17 genes at an appropriate sensitivity level in multiple patient samples. This methodology allows mutation scanning of all coding exons of 5 complete genes (TP53, EZH2, DNMT3a, RUNX1 and CEBPa) scanning of the mutational hotspots of 5 genes (ASXL1 exon 12, JAK2 exon 12, FLT3, MPL and CBL) and the analysis of specific mutation hotspots in a further 8 genes (NRAS, KRAS, NPM1, IDH1, IDH2, KIT, BRAF, JAK2 exon 14). In order to cover all these regions, a total of 99 PCR amplicons (average length 300bp range 250–400bp) were amplified for each patient sample. To avoid time consuming quantification and normalisation of individual amplicons downstream of PCR, we developed a simple method to first designate the amplicons into 4 groups based on the efficiency of PCR amplification. Following amplification the 1st round PCR products are diluted according to the grouping assigned to the particular amplicon prior to a second round of PCR in which MIDs or “barcodes” are added in a patient specific manner. The products of the 2nd round PCRs are then pooled for each patient and normalised before pooling the entire library for subsequent sequencing. The sequencing is carried out using the Roche GS FLX titanium reagents. To validate this approach we have prepared libraries from 80 AML patients with normal cytogenetics. Sequencing of these patients was split over 3 runs of the FLX instrument using standard conditions recommended by the manufacturer. The average number of reads per amplicon was 300 with 95% of amplicons having a minimum coverage of 200 reads. The approach allows the detection of mutations at a sensitivity of approximately 5%. Mutations were detected in 14 /17 genes in at least one patient sample. No mutations of BRAF, JAK2 or MPL were detected. In line with previously published data, mutations were found frequently in NPM1 (67%), DNMT3a (55%) FLT3 ITD (44%) and less frequently in the remaining genes. These data correlate well with published data for normal karyotype AML. In 6/17 genes the mutation status of the patient samples had been previously analysed using: Fragment analysis (NPM1 and FLT3 ITD), RFLP (FLT3 TKD), Sanger Sequencing (IDH1, IDH2, ASXL1, TP53). Significantly, 12 additional mutations were detected (NPM1 n=2, FLT3 ITD n=2, FLT3 TKD n=4, IDH1 n=2, IDH2 n=2, ASXL1 n=4) using the new methodology demonstrating an improvement in sensitivity over and above individual gene specific assays. A single run of the FLX instrument enables the analysis of 30 patients in a batch. In our hands library preparation, sequencing and analysis of 30 patients using the workflow described takes a skilled operator 10 full days. With an estimated test price of \P650 ($1000), this compares extremely favourably with the price of providing these analyses separately estimated to be in excess of \P4000 ($6500). To conclude we have developed a next generation amplicon sequencing approach to assay 17 individual target genes including whole gene coding sequences and more focused mutational hotspots. In addition we have validated a strategy which dramatically reduces the amount of operator time required compared with most amplicon sequencing approaches. This approach offers a highly flexible platform for analysing multiple gene targets in multiple samples. Changes to the targets analysed or the effective sensitivity can easily be incorporated without the need to make major modifications to the procedure. Disclosures: No relevant conflicts of interest to declare.

Abstract 2793 Whole Exome Sequencing Reveals Acquired SF3B1 Mutations Defining Patients with Acquired Idiopathic Sideroblastic Anaemia. Refractory anaemia with ringed sideroblasts (RARS) is an acquired form of MDS characterized by accumulation of mitochondrial iron that gives rise to ringed sideroblasts. The molecular pathogenesis of RARS and hence the cause of abnormal mitochondrial iron is unknown. To address these questions, whole exome sequencing (Illumina) was performed on CD34+ cells from 8 patients all of whom had >50% ringed sideroblasts, comprising 5 RARS, 1 RARS-T, 1 tMDS and 1 RCMD-RS patient. Following filtering against variations found in available paired constitutional samples (CD3+ T cells or skin) and published polymorphisms, candidate mutations were initially identified for commonality between patients. Aberrations in one particular gene, splicing factor 3b subunit 1 (SF3B1), a component of the major and minor spliceosomes, stood out at this stage. Mutations in SF3B1 exons 14 –16 were observed in CD34+ cells in 7/8 cases, but not in the single case of tMDS, which carried mutant TP53. Follow-up amplicon sequencing (Roche 454 and Sanger sequencing) for a total of 27 cases with >50% ringed sideroblasts, using total bone marrow tissue, identified acquired SF3B1 mutations in 93% (14/15) RARS, 67% (6/9) RCMD-RS, 100% (1/1) AML with a preceding RARS and 0% (0/2) tMDS cases. All mutations were heterozygous substitutions and found in the highly-conserved protein c-terminal HEAT motifs, associated with snRNP stabilization within the U2 snRNP complex of the major spliceosome. However, gene expression microarray analysis (Affymetrix exon arrays) showed no significant differences between cases with (n=5) and without (n=3) mutations. All the patients with SF3B1 mutations had normal cytogenetics except one patient with trisomy 8. There was no difference in overall survival or leukaemic transformation when comparing patients with or without SF3B1 mutation. Furthermore, in a single RARS case with available sequential samples, the SF3B1 mutation burden was maintained at the same level from diagnosis, at progression to AML and at time of remission following intensive chemotherapy (FLAG). SF3B1 mutations were also detected in glycophorin A + erythroid cells. Interestingly, no ‘hotspot’ SF3B1 mutations were seen in a single case of congenital sideroblastic anaemia. A possible mutual exclusivity between SF3B1 and TP53 mutations (p Exome sequencing also revealed additional novel candidate mutations, including ones in previously uncharacterized zinc finger proteins, and candidate mutations in factors which suggest defects in cell proliferation, DNA maintenance and cytoskeletal organization. In addition to known mutations mentioned above, between 30–70 well-supported candidate mutations were identified per individual exome. However, candidate mutations in the haem-synthesis pathway enzymes were rare (1/8 cases) and are therefore unlikely to contribute to the RARS disease phenotype. We therefore show that whole exome sequencing is effective in identifying novel mutations in a well-defined patient cohort. We subsequently demonstrate that mutations in the fundamental spliceosome component SF3B1, is acquired in a majority of cases of sideroblastic anemia Disclosures: No relevant conflicts of interest to declare.

Abstract 792 Myelodysplastic syndromes are clinically and molecularly heterogeneous group of disorders with variable prognosis and propensity to leukaemic transformation. We analysed the incidence and impact of TP53 gene mutations in a large cohort of MDS patients (n=318) using next generation sequencing (454 FLX). The median age was 65 years (range 17–72 years) with a male to female ratio of 1.7:1.The median follow-up of this cohort was 18.7 months (range 1–74 months). Forty patients (12%) underwent disease modifying treatments (bone marrow transplant, intensive chemotherapy, 5-azacitidine and Lenalidomide) at the time of sample collection and hence survival analysis was censored at the date of such treatment. Twenty one (7%) has RA, MDS with isolated del 5q–26 (8%), RARS-13 (4%), RCMD/RCMD-RS-105 (32%), RAEB 1/2 −75 (24%), AML secondary to MDS-29 (9%), therapy related MDS (tMDS)-22(7%) and MDS/MPN 27 (9%). IPSS cytogenetic risk groups were; good risk-199(63%), intermediate −35 (11%) and poor risk-67 (21%) and cytogenetics failed in 17 patients (5%). The IPSS categories were, low risk: 71(24%), intermediate-1:101(32%), intermediate-2:58 (18%), high risk: 29 (9%) and 38 patients were not evaluable (proliferative CMML and MPD/MDS). Progression to AML occurred in 68 patients (20%). TP53mutations were observed in 30 (9.4%) of MDS patients. Twenty of 30 (67%) mutations were detected in int-2/high risk IPSS groups and were associated with complex cytogenetics (73%). Rest of the mutations were detected in low/int-1 IPSS patients with isolated 5q- abnormality [5 of 26, (19%)].Of the 22 cases with t MDS, 6 had TP53 mutations and 17/91 (19%) patients with RAEB and AML with trilineage dysplasia harboured TP53 mutations. Only one patient each with RCMD and MDS/MPD-U had TP53 mutation. Of the 39 mutations, 9 patients had two mutant P53 clones whereas 21 patients had single mutation. 30 of 36 (83%) mutations were located in the DNA-binding domain. Patients with therapy related MDS had increased likelihood (p The median clone size of TP53 mutations were 42 %( range 2.5–93%) and the median clone size of patients with double mutations was 82%.Nine patients with TP53 mutation had sequential samples tested at variable time intervals post 5-azacitidine treatment to analyze clone size and correlate with clinical responses. Four patients showed a marked decrease in clone size (24 to 3%,25:44% to 4:6%,35; 40% to 8; 17%78% to 15%,) associated with cytogenetic remission, one patient showed a increase in clone size (60% to 93%) at the time of leukaemic evolution and the remaining four patients had stable TP53 clones after 5-azacitidine. TP53 mutations strongly correlated with worse OS and PFS. The median OS for patients with wild type TP53 was not reached (NR,>66 months) compared with 9.7 months for patients with TP53 mutations (p66 months) compared with 8.5 months for patients with TP53 mutations (p A cytogenetic sub-group analysis was performed in patients with complex cytogenetics with or without mutations of TP53, and showed patients with mutation had a worse OS (9.6 vs. 14.1 months, p MDS with isolated deletion 5q harbouring a TP53 mutation(med OS 23 months) had significantly worse outcome compared with del5q patients without mutation(med OS 66 months)(p In a multivariate analysis using co-variables: age (>55yr vs This large single institution study confirms that TP53 mutations are an independent prognosticator of outcome in MDS. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating the prognosis in patients with MDS. The relatively common occurrence of these mutations in different spectrums of MDS, ie isolated 5q- and complex cytogenetics, possibly implies two different mechanistic roles for p53 protein in disease evolution. Disclosures: Elebute: Alexion: Honoraria. Mufti:Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 1340 We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response. Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures: No relevant conflicts of interest to declare.

Abstract 125 Polycomb gene complex mutations have been implicated in the pathogenesis of myeloid neoplasms. ASXL1, a epigenetic regulator of transcription by recruiting polycomb and trithorax complexes to the chromatin domain and EZH2 (histone methyltransferase) is the catalytic domain of polycomb repressive complex (PRC) 2, causing gene silencing by trimethylation of lysine 27 of histone 3(H3K27). To identify the role of mutations in ASXL1, EZH2 and their interaction with other mutations (P53, ras, c-cbl, IDH 1/2, BRAF), we analysed 63 MDS patients treated with 5-Azacitidine (Aza) at our institution. Mutation analysis was done by deep (454 FLX) and Sanger sequencing. SNP-6 karyotyping was also performed to correlate with mutation status. In 43 patients, samples were also analysed at various time points post aza treatment. The median age was 67 years (range 36–86 years), median number of courses 7(range 2–109), 90% (57/63) of patients belonged to high risk IPSS category. WHO category subtypes were; RA/RARS-1; RCMD-7; RAEB-37; s-AML (evolved from pre-existing MDS) -9, therapy related myeloid neoplasm (t-MDS/t-AML) -7 and CMML-2.IPSS cytogenetic subgroups were, good risk: 19, intermediate: 6, and poor risk: 38. Thirty six patients had chromosome 7 abnormalities either isolated (12/36) or with additional aberrations. The median time from diagnosis to treatment with aza was 7 months (range 1–42). The overall response rate to Aza was 48% (30/63) with a better survival in responders compared with non-responders (17.3 vs 10.2 months p ASXL1 exon 12 mutations were present in 22/63 patients (35%), good risk cytogenetics (8/19), intermediate risk (4/6), isolated monosomy 7 patients (7/12) but none in the complex cytogenetics subgroup (p P53 mutations were detected and quantified by high throughput sequencing in poor risk IPSS cytogenetic group (14/38) and were absent in patients with normal cytogenetics and isolated chromosome 7 abnormalities. EZH2 mutations were not detected in patients with P53 mutations and only 2/22 patients with ASXL1 had a concurrent P53 mutation, suggesting a possible mutual exclusivity of polycomb gene mutations and P53. All 14 patients with P53 mutation died (p IDH1and IDH2, C-Cbl, NRAS mutations occurred in 6/63 (10%), 4/63 (6%), 6/63 (10%) of patients respectively and did not correlate with clinical outcome. Age, IPSS cytogenetic risk group, WHO subtypes, overall response rate (ORR),progression to AML, P53 mutation status and presence of EZH2 and/or ASXLI mutation were used as co-variables in multivariate analysis.P53 mutation status (HR=5.3(2.3-11.8), p This study shows that polycomb complex gene mutations are frequent particularly in relatively good cytogenetic subgroups of MDS. The presence of these mutations correlates with a significantly better OS and PFS following Aza therapy. P53 mutations are uncommon in patients that harbour polycomb complex gene mutations and the presence of P53 mutations do not correlate with response rate, however they remain a strong indicator of OS and PFS. Disclosures: No relevant conflicts of interest to declare.

Abstract 733 Tet2 is a novel candidate tumor suppressor and mutations in this gene have been found in 24% of myeloid neoplasms following conventional Sanger sequencing. Using a massively parallel pyrosequencing methodology (Roche GS FLX) we sequenced the entire Tet2 gene coding region in 360 patients with myeloid malignancies (MDS 279; CMML 32; AML 10; MDS/MPD 10; 5q syndrome 8; others 21). We also utilized high density SNP6 arrays to screen for chromosomal aberrations including those at the Tet2 locus itself. Initially we found 106 mutations in Tet2 corresponding to 75 patients (21%), where the average sequence coverage was more than 100-fold for >90% of the gene. 25 patients had more than one mutant Tet2 clone. 83 mutations (in 17% of patients) were found in 30% to 95% of sequenced clones, where 50% or 100% could be indicative of heterozygous and homozygous mutations that would be found in all sample bone marrow cells respectively. The remaining 23 mutations were found in ≤30% of sequence clones, applying a lower threshold of 17% to avoid false positives, and were not reliably picked up by confirmatory Sanger sequencing. The 106 mutations were distributed as 44 nonsense or InsDel mutations throughout the gene, 59 nonsynonomous mutations within (23) and outside (36) the two known conserved domains, and 3 spice site mutations. Tet2 mutations were not correlated with survival outcome (probability of survival after 3 and 5 years from diagnosis 40% and 38% [p=0.3 log rank]for mutants vs non-mutants respectively). Patients with small (≤30%) or multiple mutant clones did not deviate from the overall survival trend. Furthermore, no difference in survival was seen when patients were analysed according to their WHO subgroups or IPSS scores. CMML featured as the only subgroup with a relatively high Tet2 mutation rate (53% of patients). Chromosomal aberrations linked to the Tet2 locus, including UPD or micro-deletion, were detected in 20-25% of patients, but this was not strongly correlated to detectable mutations in the Tet2 gene itself (24% coincidence), perhaps indicating that several forms of mutation or instability at this region are linked to disease. Interestingly, high percentage mutant clones which are indicative of homozygosity and thus loss of heterozygosity (LOH), were found as InsDel mutations in Tet2 for the majority (85%), suggesting a common defect in DNA replication or repair. Following up on additional smaller Tet2 mutant clones, we lowered our threshold for mutation detection and after further analysis and removal of experimental background, 42 additional candidate mutations of clone size between 8% and 17% were found. Taken as a whole, Tet2 gene mutations were found in 30% of patients but this may rise considerably if deeper sequencing screens for even smaller mutant clones yield further positive results. We therefore show via highly sensitive ‘Deep sequencing' on a relatively large patient cohort a comprehensive and highly quantitative mutation map across the Tet2 gene to allow increased prognostic comparisons. We also show that mutation figures rise significantly when smaller mutant clones are considered that cannot be detected via Sanger sequencing. In addition to this, if larger chromosomal aberrations such as UPD are added to the evolving number of mutant Tet2 patients, then Tet2 abnormalities may in fact be present in a majority of patients with myeloid neoplasms. Disclosures: Gattermann: Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.