ALBERT

Description: 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

Description: 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

Description: 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

Description: The ability to impose exogenous targeted epigenetic changes in the genome represents an attractive goal in gene therapy for the heritable repression of target genes, while potentially enabling the generation and subsequent study of the downstream effects of de novo epigenetic events, which are known to occur in disease. Here we demonstrate the ability of zinc-finger peptides to deliver DNA cytosine methylation in vivo to a genomic target promoter, when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular de novo methylation events. We show for the first time targeted gene silencing in response to directed DNA cytosine methylation via initiation of a repressive chromatin signature at a targeted genomic locus, characterised by elevation of histone H3K9Me2 and reduction of H3K4Me3 levels at that region. This transcriptional repression is maintained in the absence of sustained targeted methyltransferase action, confirming epigenetic maintenance by the cells own machinery. The inherited DNA methylation pattern is restricted to specific target sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. Therefore, we demonstrate for the first time, targeted DNA methyltransferases as potential tools for the exogenous and heritable control of gene expression at the chromosomal level, while providing the clearest and most direct confirmation to date of the functional and mechanistic consequences of de novo DNA methylation in the cell. This work represents an important step towards the longer term goal of controlling gene expression through the inheritance of a repressive DNA state, as well as providing a valuable tool for studying spatial and temporal issues associated with ‘genuine’ de novo methylation, on transcription and chromatin structure.

Description: p73 is a member of the p53-family of transcription factors (p73, p53 and p63). A truncated form of p73 (ΔNp73) also exists that lacks the transcriptional activation domain and inhibits both p53 & p73. However, ΔNp73 transcripts have not been reported in normal tissues or peripheral blood. We now show for the first time that ΔNp73 mRNA and protein are not present in quiescent (G0), primary human T cells but are induced post the G0→G1 cell cycle commitment point following stimulation with anti-CD3/CD28 or PMA/ionomycin. The ΔNp73 transcript could be produced either by activation of a promoter in intron 3 or from the 5′ P1 promoter by alternative splicing. Bisulfite sequencing shows that the intron 3 promoter is hypermethylated in T cells in G0 and throughout the cell cycle while the 5′ P1 promoter remains largely unmethylated. 5′-RACE results with quiescent and stimulated T cells verify that ΔNp73 transcripts originate from the P1 promoter. Additionally, RT-PCR analyses show that the transcript originating from the P1 promoter is present in G0 and mature ΔNp73 mRNA is produced by cell cycle-dependent splicing during the G0→G1 transition. We investigated the function of ΔNp73 during G0→G1 by inhibiting its induction with two different siRNAs. Microarray analyses show that expression of mRNA encoding a number of cell cycle regulators like E2F1, TCFL2 and CDT1 during G0→G1 are dependent on ΔNp73 and ΔNp73 represses the expression of the pro-apoptotic PUMA, a known p73 target. We conclude that ΔNp73 is produced in normal, human T cells during cell cycle entry and regulates normal gene expression. Moreover, the intron 3 promoter is hypermethylated, the ΔNp73 transcript originates at the P1 promoter and ΔNp73 mRNA is produced by cell cycle-dependent splicing.

Description: 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= 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.

Description: Introduction: Hypocellular myelodysplastic syndrome (hMDS) is characterized by decreased marrow cellularity, and is often difficult to distinguish from aplastic anemia (AA) based on standard morphological criteria. It represents around 10-15% of patients diagnosed with MDS, but is not currently considered a separate entity by WHO. Hypocellularity is defined as bone marrow cellularity of less than 30% in patients younger than 70 years or less than 20% in those older than 70 years. Patients and Methods: We retrospectively evaluated the demographic, clinical features, bone marrow aspirate/trephine, treatment characteristics and outcomes of 100 patients with hMDS. The bone marrow aspirates and trephines were also analysed for dysplasia, blast percentage, cellularity, reticulin, p53 by Immunohistochemistry (IHC), CD34 and CD117. An MiSeq based targeted gene panel comprising of 24 frequently mutated MDS genes was used in a subset of patients. Results: The median age was 51 years (18-87), with only a third of patients 〉60 years. WHO subtypes RA (n=2), MDS 5q- (n=1), RCMD (n=95) and RAEB (n=2), IPSS risk groups low risk (n=20), Int1 (n=61), int2 (n=11) and high risk (n=2). 23% had evolved from previous AA. For the hMDS group IPSS cytogenetic categories comprised good risk 67% (n=62), intermediate 14% (n=13), including 6 cases of trisomy 8, and poor 18% (n=17), with 7 cases of monosomy 7. Of the normal cytogenetics, 15/62 (24%) had positive (moderate to strong) p53 by IHC, 30/62 (48%) with CD117 positivity (1-20%), 48/62 (77%) have fibrosis grade 1-2. Only 2/62 with normal cytogenetics had normal p53, CD34, CD117 and reticulin and these have evidence of dysplasia on morphology. The presence of cytogenetic abnormalities and other features such as p53, CD117 and fibrosis reflect a distinct population differing from the hypocellular marrow of aplastic anemia. A higher incidence (18%) of autoimmune disorders was seen, including ITP (n=2), thyroid dysfunction (n=3), alopecia (n=2), inflammatory bowel disease (n=4), coeliac (n=2), SLE/SjogrenÕs (n=2), others (n=6). Forty percent (n=32) had a PNH clone, all except 2 were subclinical. Progression of disease to AML (n=3), upstaging of disease (RCMD to RAEB, n=6) or cytogenetic evolution (n=3) was seen in 15% of hMDS, including 3 cases with both increase in blasts and karyotypic evolution. A subset (n=33) of hMDS were evaluated for recurrently mutated genes in MDS; 7/33 (21%) of patients harbored somatic mutations in ASXL1 (n=4), DNMT3A (n=2) and BCOR (n=2). All except one patient with somatic mutation had a prior history of aplastic anaemia. As the predominant group was hypocellular RCMD (95%), we compared the clinical features, treatment and prognosis with cellular RCMD cohort during the same time period. Median ages of hMDS and non-hypocellular MDS (nhMDS) were 51 and 60.3 years respectively. Patients with hMDS presented with more significant thrombocytopenia (median platelet count at presentation 43 vs 93), neutropenia (median ANC at presentation 1.13 vs 1.3), anaemia (median Hb 94g/L vs 104g/L), transfusion dependency, and more intermediate-2/high-risk disease than the nhMDS group (p=0.0257). Among hMDS patients, 30% (28/95) had chromosomal abnormalities, an incidence similar to that of nhMDS, 25% (23/94). Treatments received by hMDS cohort were: single agent Cyclosporin (n=27), anti-thymocyte globulin (n=7), erythropoietin and GCSF (n=13), 5-azacytidine (n=6), intensive chemotherapy (n=4), HSCT (n=26), including 9 patients who underwent upfront HSCT. As expected the use of IST was infrequent (8%) in the nhMDS cohort compared to 35% in the hMDS (p

Description: Ischemic stroke is a devastating complication of sickle cell anemia (SCA) and can affect children from a very young age. It is the commonest cause of stroke in the general pediatric population. The peak incidence is in the first decade of life and approaches 11% in the absence of stroke prevention programmes. The advent of transcranial doppler (TCD) screening as part of a primary stroke prevention programme has reduced the incidence of stroke by 90%. However, not all strokes are prevented by TCD, and TCD abnormalities are not specific, with up to 60% children receiving transfusions unnecessarily. Understanding the underlying pathophysiology of cerebrovasculopathy in SCA and recognition of important risk factors will lead to a more stringent identification of the at-risk population. Analysis of siblings with stroke and abnormal TCDs offers good evidence that a proportion of this variability is conferred by inherited genetic variation. Identifying those specific variants has proved elusive, with many conflicting findings reported in the literature, no doubt reflecting the limited power of many studies to address the complex overlay of environmental and genetic influences. To attempt to minimise the impact of non-genetic confounders, our study looked specifically at children who had an overt ischemic stroke or developed abnormal TCD measurements (TAMMV 〉200cm/s) prior to their 4th birthday, in the absence of potentially precipitating acute medical events. We recruited 22 patients, 19 of whom had an ischemic stroke, and 3 who developed abnormal TCD measurements, prior to 4 yrs of age. Additionally, we recruited the dizygotic twin of one stroke case, who had SCA, but, importantly, no cerebrovascular complications, as confirmed by MRI/A aged 15 yrs. We extracted DNA from peripheral blood samples. Exome library was prepared using Agilent SureSelect XT V7, sequencing was performed using Illumina NovaSeq. Alignment, assembly, variant calling and annotation were based on a GATK Best Practices workflow. For each sample, around 15000 non-synonymous variants were identified. Across the 22 case samples, 58 variants in 56 genes were predicted to be pathogenic or likely pathogenic, as determined with InterVar. These variants were interpreted with respect to dbSNP documentation and biological role of the gene they affected. Variant segregation within the twin pair was also considered. Two patients were homozygous for rs429358, a missense variant in the APOE gene that is pathogenic for familial hyperlipoproteinemia, type 3. Moreover, 3 further patients were compound heterozygous for this variant plus another APOE missense variant pathogenic for the same condition, rs7412. One of these 3 cases was the affected sibling of the twin pair and notably, the unaffected twin carried only heterozygous rs429358 and not rs7412. A further patient was heterozygous for rs121908043 in the LDL-R gene, which is pathogenic of Familial Hypercholesterolaemia, even in the heterozygous state. Finally, in an additional patient, we identified a potential compound heterozygote of a known pathogenic variant, rs118204068 with another missense variant rs11542065 in the LPL gene, to cause Hyperlipoproteinemia type 1. We have used whole exome sequencing to analyse patients with sickle cell anemia and stroke at a very young age, an extreme phenotype, in whom we predicted genetic factors would be a significant cause of stroke. We found 7 of the 22 patients (32%) had variants diagnostic of inherited dysplipidaemias, including 5 with familial hyperlipoproteinemia type 3, 1 with hyperlipoproteinemia type 1 and one with familial hypercholesterolemia. Our analysis included one twin set and it is noteworthy that the unaffected twin sibling did not carry the necessary variants defining the inherited dyslipidaemia. These conditions are characterised by improper breakdown and accumulation of LDL-C, triglycerides and cholesterol, which has been associated with vascular dysfunction and hemolysis in SCA and are strongly associated with stroke and cardiovascular disease in the general population. This is potentially an important finding that warrants further investigation into the role of lipids and hyperlipidemia in the development of stroke and cerebral vasculopathy in the sickle cell population. In the long term, we suggest that measurement, and potentially, control of lipids may form a component of the primary stroke prevention programme. Disclosures Brousse: Add medica: Consultancy; bluebird bio: Consultancy. Rees:Novartis: Other: Strategic advisory role,Principal investigator,sickle cell disease2.6 Investigator; Astra Zeneca (ticagrelor): Other: Data monitoring committees; TauRx (methylene blue): Other: Data monitoring committees; Alnylam: Other: Principal investigator; Global Blood Therapeutics: Other: Strategic advisory role; Celgene: Other: Strategic advisory role; Emmaus: Other: Strategic advisory role; Agios: Other: Grants.

Description: Key Points Acquired mutations of myeloid-related genes are present in a proportion of AA patients. Somatic mutations in AA predict higher risk of transformation to MDS.