ALBERT

Description: Abstract 171 The 5q- syndrome is the most distinct of all myelodysplastic syndromes with a clear genotype/phenotype relationship. Haploinsufficiency of the ribosomal protein gene RPS14 underlies the erythroid defect found in the 5q- syndrome. The 5q- syndrome is a disorder of aberrant ribosome biogenesis and we have recently demonstrated that erythrocytes obtained from patients with the 5q- syndrome show impaired translation. This defect in translation may represent a potential therapeutic target in the 5q- syndrome and other ribosomopathies, and there are some indications that the use of the translation enhancer L-leucine may have some efficacy. L-leucine is a branched chain amino acid that has been shown to improve haemoglobin levels and transfusion independence in patients with the ribosomopathy Diamond Blackfan Anemia (DBA). Moreover, the treatment of zebrafish models of DBA and the 5q- syndrome with L-leucine has recently been shown by others to result in partial reversal of the anemia. To model the RPS14 haploinsufficiency observed in the 5q- syndrome, we used lentivirally delivered shRNA sequences to reduce the expression of RPS14 in human bone marrow CD34+ cells from healthy controls. We have recently shown that treatment of cultured human erythroid cells derived from CD34+ cells of healthy controls with RPS14 knockdown and cultured erythroid cells derived from the CD34+ cells of patients with the 5q- syndrome with L-leucine results in an increase in cell proliferation, erythroid differentiation and mRNA translation. There is evidence to suggest that L-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls many cellular processes including cell growth and mRNA translation. In order to investigate the mechanism of action of L-leucine, we have studied the phosphorylation levels of S6K1 and 4EBP1, the key downstream targets of mTORC1 (mTOR Complex 1), by sandwich ELISA, Human Phospho-kinase Antibody Array and flow cytometry in RPS14-deficient human erythroblasts. We have shown a significant increase in the levels of phospho-S6K1 and phospho-4EBP1 following L-leucine treatment of cultured erythroid cells derived from CD34+ cells of healthy controls with RPS14 knockdown. The effects of L-leucine on phospho-S6K1 and phospho-4EBP1 were abrogated by rapamycin (an mTOR inhibitor), suggesting that L-leucine has a specific action on the mTOR signaling pathway. Consistent to the results observed in the RPS14 knockdown model, treatment with L-leucine also increased the level of phospho-S6K1 in cultured erythroid cells derived from the bone marrow cells of 5q- MDS patients. These data suggest that L-leucine activates the mTOR pathway in RPS14-deficient human erythroblasts. Our studies support the evaluation of L-leucine as a potential therapeutic agent in the treatment of the 5q- syndrome, and provide evidence on its mode of action through activation of the mTOR signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Approximately 30-40% of patients with myelodysplastic syndromes (MDS) develop acute myeloid leukemia (AML). Several recurrent mutations have been identified in MDS using next-generation sequencing (NGS) technology and recent studies have greatly illuminated the molecular landscape of this disorder. However, the molecular events driving MDS progression to AML remain poorly understood. In order to investigate the genetic basis of leukemic transformation in MDS during disease progression, we evaluated the frequency and chronology of the acquisition of mutations using a targeted NGS myeloid gene panel on serial (paired) samples from 41 MDS patients before (pre-progression) and after disease progression (post-progression) to a more advanced subtype (n=7) or to AML (n=34). The mutational profile was characterized using a TruSeq Custom Amplicon panel (Illumina) targeting the hotspots of 31 recurrently (〉1%) mutated genes in myeloid malignancies. Samples were run on an Illumina MiSeq and variants were annotated and filtered using Illumina VariantStudio v2.1.36, and interpreted according to the ACMG recommendations. The proportion of sequencing reads reporting a given mutation (variant allele frequency, VAF) was used to estimate the fraction of tumor cells carrying that mutation, and to determine whether mutations are clonal (in all tumor cells) or subclonal (in a fraction of tumor cells). A total of 100 and 123 mutations across 23 genes were identified in pre- and post-progression samples, respectively. The number of mutations was generally higher in the post-progression samples: the number of cases with one or two mutations was 24 in pre-progression samples and 16 in post-progression samples, while the number of cases with three or four mutations was 12 in pre-progression samples and 18 in post-progression samples. Several cases showed ≥5 mutations. The most frequently mutated genes (in 〉15% of samples) were ASXL1, TET2, SRSF2, U2AF1, RUNX1 and TP53; ASXL1 was the top ranking mutated gene with a frequency of 44% in pre-progression samples and 46% in post-progression samples. SF3B1, the most frequently mutated gene in MDS, was mutated in only two cases in our cohort. This finding is consistent with the strong association of SF3B1 mutation with the low-risk MDS subtype RARS, and its status as a good prognostic marker. Our patient cohort is highly selected, comprised only of patients whose disease has progressed and our data thus indicate that ASXL1 mutations are strongly associated with MDS cases that show disease progression and conversely that SF3B1 mutations are rarely associated with MDS disease progression. Mutations of genes involved in splicing (U2AF1, SRSF2), chromatin modification (EZH2, ASXL1) and DNA methylation (TET2, IDH1/2) were present in the pre-progression samples for almost all cases with mutations of these genes; these mutations often occur in founding clones (VAF 〉40%) and may play a role in disease initiation. Mutations of genes involved in transcriptional regulation (RUNX1, ETV6, PHF6) and signal transduction (NRAS, KRAS) were found in many cases in the post-progression sample only, suggesting that these are often late events that may co-operate with early events to drive disease progression. Interestingly, co-occurrence of NRAS and ASXL1 mutations was a frequent event in post-progression samples (n=5) in our cohort. It has been reported that NRas mutation and Asxl1 loss co-operate to drive myeloid proliferation and myeloid leukemia in mice, and our data support this observation. The average VAF of some mutations changed markedly during disease progression, with NRAS showing the largest VAF fold increase among genes mutated in 〉5 cases. The majority of subclones containing mutations in RUNX1 or ASXL1 expanded with disease progression. EZH2 mutations were mainly in the founding clone, while TP53 mutations were mainly in a subclone. For six cases we sequenced an additional serial sample, thus giving more precise information on the mutational profile evolution during disease progression. This is the first study to investigate the mutation status of a large group of MDS patients showing disease progression by the study of serial samples using a NGS myeloid gene panel. This study informs the timing of mutation acquisition during disease initiation and progression in MDS and closely related conditions, illuminating the genetic basis of leukemic transformation in these disorders. Disclosures No relevant conflicts of interest to declare.

Description: The recent discovery of a variety of somatic splicesomal mutations in the myelodysplastic syndromes (MDS) has revealed a new leukaemogenic pathway involving spliceosomal dysfunction. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP proteins. The snRNPs are involved in recognising short conserved sequences of the pre-mRNA, including the 5′ and 3′ splice sites and the branch site, and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. Mutation of the Splicing Factor 3B, subunit 1 (SF3B1) gene is common in MDS, occurring in over 70% of patients whose disease is characterised by ring sideroblasts (RARS). The close association between SF3B1 mutation and ring sideroblasts is consistent with a causal relationship, and makes this the first gene to be strongly associated with a specific feature of MDS. Sf3b1 heterozygous knockout mice show the presence of ringed sideroblasts. In order to investigate the role of SF3B1 haploinsufficiency in MDS we have silenced SF3B1 using siRNA in the myeloid cell lines K562, TF-1, SKM1, HeL and OCIM2. Cell growth was impaired in all the cell lines with SF3B1 knockdown. Using Flow Cytometry, cell cycle analysis showed a significant increase in cells in the sub-G0 phase as well as G2/M arrest in the cell lines. We also observed impaired erythroid differentiation in hemin treated K562 and TF-1 cell lines with SF3B1 knockdown. Gene expression profiling (GEP) was performed in two cell lines with SF3B1 knockdown (K562 and TF1). Deregulated pathways and gene ontology categories included cell cycle regulation and alternative splicing using Ingenuity Pathway Analysis. We next performed Gene Set Enrichment Analysis (GSEA). The GSEA showed a significant enrichment of nonsense-mediated mRNA decay (NMD) genes that were up-regulated in cells with SF3B1 knockdown, suggesting NMD activation following SF3B1 silencing. We used Human Exon-Junction arrays (Affymetrix) to evaluate global transcript exon usage in the K562 and TF1 cell lines with SF3B1 knockdown. We observed significant differential exon usage in genes involved in RNA degradation, spliceosome, cell cycle and apoptosis. We further observed aberrant splicing of the candidate gene ABCB7 showing exon skipping and TP53 gene showing exon skipping as well as intron retention. We have investigated the changes in the transcriptome in CD34+ cells from MDS patients with SF3B1 mutation by RNA sequencing and found many genes showing significant differential exon usage including CCND1, EIF3B, FKBP1A, BCL2 and RB1. Using Ingenuity Pathway Analysis we identified alternative splicing pattern of genes involved in cell cycle, RNA processing, mTOR signalling and P53 signalling pathways. We have studied CD34+ cells from MDS patients with SF3B1 mutation in vitro and observed impairment in cell growth compared to CD34+ cells from healthy controls or from MDS patients without splicing mutations. In colony forming assays we observed a decrease in the number of erythroid or myeloid colonies derived from CD34+ cells of patients with SF3B1 mutation compared to patient CD34+ cells without splicing factor mutation. The identification of SF3B1 downstream targets in SF3B1 mutant and wild-type erythroid and myeloid colonies from MDS patients is in progress using RNA sequencing. Our results show that knockdown of SF3B1 in haematopoietic cell lines results in impaired cell growth, deregulated global gene expression and aberrant splicing. Studies of the haematopoietic progenitor CD34+ cells of patients with SF3B1 mutation show impaired cell growth and erythroid differentiation as well as deregulation of many pathways including the cell cycle and RNA processing. The identification of the key target genes affected by the common splicing mutations in MDS is critical to our understanding of how the mutations contribute to the pathogenesis of this disorder. Disclosures: Maciejewski: NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Description: Extracellular vesicles (EVs) are membrane-bound particles involved in intercellular communication. They carry proteins, lipids, and nucleotides such as microRNAs (miRNAs) from the secreting cell that can modulate target cells. We and others have previously described the presence of EVs in peripheral blood of multiple sclerosis (MS) patients and postulated them as novel biomarkers. However, their immune function in MS pathogenesis and the effect during the onset of new immunomodulatory therapies on EVs remain elusive. Here, we isolated plasma EVs from fingolimod-treated MS patients in order to assess whether EVs are affected by the first dose of the treatment. We quantified EVs, analyzed their miRNA cargo, and checked their immune regulatory function. Results showed an elevated EV concentration with a dramatic change in their miRNA cargo 5 h after the first dose of fingolimod. Besides, EVs obtained prior to fingolimod treatment showed an increased immune regulatory activity compared to EVs obtained 5 h post-treatment. This work suggests that EVs are implicated in the mechanism of action of immunomodulatory treatments from the initial hours and opens a new avenue to explore a potential use of EVs for early treatment monitoring.

Description: Abstract 2940 Recent studies have identified mutations of the ASXL1 gene in MDS and CMML. Mammalian ASXL proteins are believed to play a role in chromatin remodelling. We have previously reported that ASXL1 mutations are frequent in patients with MDS, CMML and AML. We observed that the mutation rate was relatively high in late MDS and AML with a lower frequency across early MDS. We found that the mutation is common in the normal karyotype group (especially AML secondary to MDS), occurring in 40% of all cases. ASXL1 mutations (including the common mutation c.1934dupG;p.Gly646TrpfsX12) were present in myeloid cells, but not in T-cells, indicating that they were acquired in all seven cases examined. The majority of the mutations identified were heterozygous frameshift mutations caused by deletion or duplication of a nucleotide. Given the high frequency of ASXL1 mutations in advanced MDS and AML in our earlier study, we have now screened a larger group of 143 AML samples, comprising 111 de novo AML and 32 AML secondary to either MDS or CMML, for mutations in the ASXL1 gene. In primary AML samples we found only 6/111 patients with ASXL1 mutations (5%), whereas the frequency of mutations was significantly higher in AML secondary to MDS (10/25, 40%) or CMML (5/7, 71%), strongly suggesting an association with disease progression in MDS and CMML. In order to determine the frequency of ASXL1 mutations in de novo and secondary AML patients with a normal karyotype and to identify cooperating mutations, we screened 85 samples for ASXL1 (all coding exons), NPM1 (exon 12), FLT3 (ITD and D835Y), TET2 (all coding exons), IDH1 (R132), IDH2 (R140 and R172) and RUNX1 (exons 3–7) mutations (Table 1). FLT3 and NPM1 mutations were more common in de novo AML (FLT3 51% and NPM1 58%) than in sAML (FLT3 9% and NPM1 12%), consistent with the role of NPM1 mutations as a hallmark of cytogenetically normal de novo AML. TET2 was mutated in 13% of primary AML, and in 28% of sAML, with most TET2 mutations found in AML secondary to CMML (5/7, 71%), as expected according to previous reports on higher mutation frequency in CMML patients. Mutations in IDH1 and in IDH2 were evenly distributed in primary and secondary cases. Similarly, RUNX1 mutation rate showed no significant differences between primary and secondary AML cases. Interestingly, ASXL1 mutations were mutually exclusive with NPM1 mutations, suggesting that they could be markers of different subgroups with a distinct aetiology. In conclusion, we have shown that mutation of ASXL1 is a very frequent event in AML secondary to MDS and CMML, but is much less frequent in de novo AML. Our data support a role for ASXL1 mutations in disease progression in MDS and CMML. Table 1. Mutation rate for genes screened in AML samples Primary AML Secondary AML From MDS From CMML ASXL1 5/53ü(9.4%) 10/25ü(40%) 5/7ü(71.4%) NPM1 31/53ü(58.5%) 3/25ü(12%) 1/7ü(14.3%) FLT3 26/51ü(51%) 3/25ü(12%) 0/7 TET2 7/52ü(13.5%) 4/25ü(16%) 5/7ü(71.4%) IDH1 7/52ü(13%) 3/25ü(12%) 0/7 IDH2 7/52ü(13%) 2/25ü(8%) 1/7ü(14.3%) RUNX1 6/53ü(11.3%) 4/25ü(16%) 1/7ü(14.3%) Disclosures: No relevant conflicts of interest to declare.

Description: Myeloid neoplasms (MN) are usually sporadic late-onset cancers; nevertheless, growing evidence suggests that ~5% of the cases could emerge as a consequence of inherited predisposition. Indeed, the 2016 revision of the World Health Organization (WHO) includes hereditary myeloid malignancies (HMMs) as a separate disease entity. Distinguishing somatic from germline variants is of vital importance, in order to establish an appropriate individualized management and counsel the patients and their relatives. Germline tissues are required to discriminate HMMs alterations, but they are not always available for testing at the time of diagnosis; therefore additional strategies should be put in place in order to identify variants potentially conferring genetic predisposition to MN. The aim of the present work is to develop a tool to identify HMMs cases from tumour-only sequencing data. To this end, we reviewed tumor-only NGS reports from 299 cases of patients with myeloid malignancies sequenced with a custom Pan Myeloid Panel (PMP) that targets 48 genes, of which 21 are described in literature as HMM-associated genes. All patients signed a written informed consent form for genetic testing, research and tissue banking provided by the Biobank of the University of Navarra (UN) and were approved by the Ethical and Scientific Committees of the UN. Variant calling files (VCF) of the 299 cases were run through an algorithm aiming at identifying those variants suggestive of being of germline nature (Figure 1). We considered as indicative of potential inherited origin, variants detected in bone marrow samples at a ~50% VAF classified as pathogenic, likely pathogenic or of unknown significance. This first filter yielded 90 "likely HMMs" patients harboring a total of 104 suspicious of being germline variants affecting at least one of the 21 HMM-associated genes. Our algorithm included three additional filters: mutational patterns suggestive of germline nature of the variants, analysis of variant allele frequency (VAF) on follow up data, and sequencing of non-myeloid tissues (Figure 1). Firstly, we found 10 "likely HMMs" patients presenting double hit mutations in one of three genes: DDX41 (n=7), CEBPA (n=2) and RUNX1 (n=1). Additionally, there was available follow up data for 8 cases included in our cohort, harboring 11 suspicious variants. From them, VAFs in 4 variants affecting ASXL1, CBL, IKZF1 and GATA2 genes, drastically changed over time, indicating that those variants were somatic, whereas the other 7 variants affecting ASXL1 (n=2), DDX41 (n=2), IKZF1 (n=1), and RUNX1 (n=1) genes suspected to be germline maintained VAF ~50% with evident change of the accompanying variants over time. Finally, we collected non-myeloid tissue samples from 8 patients, harboring a total of 9 variants in ASXL1 (n=2), DDX41 (n=1), GATA2 (n=1), IKZF1 (n=1), NF1 (n=3), and SH2B3 (n=1) genes. Sequencing data confirmed germline nature of 6 of the 9 tested variants; 3 of the 6 germline variants have previously been shown to be linked to MN (DDX41 p.Asp140Glyfs*2 in UPN1, ASXL1 p.Gly967del in UPN11, and NF1 p.Met992del in UPN19). Of note, two of these bona-fide pathogenic germline mutations had been previously highlighted as potential germline variants when applying our criteria of mutational patterns and/or follow up data indicative of germline nature, in so showing the effectiveness of our algorithm (DDX41 in UPN1, and ASXL1 in UPN11). Regarding the usefulness of the different tissues, we found that skin fibroblasts, hair follicles and CD3+ T cells helped discriminating the nature of the variants; on the contrary, DNA from hair follicles showed poor DIN values and scarce DNA concentration. Similarly, DNA isolated from buccal swab showed in general poor quality metrics, and high level of contamination with tumour DNA in one case, proving it unsuitable for discrimination of the nature of the variants in some instances. Our data supports the importance of considering variants found upon tumor-only sequencing as potentially of germline origin, and we offer a pipeline to define the nature of the variants. We hope to provide insights for genetic laboratories facing this relatively new challenge of discriminating somatic from germline variants in tumor sequencing data. Disclosures No relevant conflicts of interest to declare.

Description: Follicular lymphoma (FL) is a common indolent B-cell lymphoma that can transform into the more aggressive transformed FL (tFL). However, the molecular process driving this transformation is uncertain. In this work, we aimed to identify microRNA (miRNA)-binding sites recurrently mutated in follicular lymphoma patients, as well as in transformed FL patients. Using whole-genome sequencing data from FL tumors, we discovered 544 mutations located in bioinformatically predicted microRNA-binding sites. We then studied these specific regions using targeted sequencing in a cohort of 55 FL patients, found 16 recurrent mutations, and identified a further 69 variants. After filtering for QC, we identified 21 genes with mutated miRNA-binding sites that were also enriched for B-cell-associated genes by Gene Ontology. Over 40% of mutations identified in these genes were present exclusively in tFL patients. We validated the predicted miRNA-binding sites of five of the genes by luciferase assay and demonstrated that the identified mutations in BCL2 and EZH2 genes impaired the binding efficiency of miR-5008 and miR-144 and regulated the endogenous levels of messenger RNA (mRNA).