ALBERT

Description: Background and aim: Treatment with erythropoietin stimulating agents (ESA) is first-line treatment for anemic low-risk MDS patients, but although clinical variables such as endogenous serum erythropoietin levels have been associated with response, the bone marrow (BM) cell populations targeted during ESA-induced erythroid improvement have not been identified. Initiating SF3B1 mutations in MDS-RS arise in the multipotent hematopoietic stem cell (HSC), and we previously showed that most SF3B1-mutated MDS-RS patients retain a small residual wildtype HSC population, something that we propose may have clinical relevance (Mortera-Blanco, et al, Blood 2017). In this study, we aimed to map clonal dynamics of mutant and wildtype hematopoietic stem and progenitor cells (HSPCs) during steady-state and ESA treatment. Methodology: We used advanced flow cytometry to sort and characterize HSPC populations in BM samples of SF3B1-mutated MDS-RS patients with stable anemia, before and during ESA response, and during chronic transfusion dependency. In addition to conventional HSPC markers, we added CD105 to the antibody panel as it has been reported to distinguish committed erythroid progenitors (Mori, et al, PNAS 2015). Variant allele frequency (VAF) of the known SF3B1 mutations was determined using droplet digital PCR. Results: Expression patterns of mature CD38+ progenitors; common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and megakaryocytic-erythroid progenitors (MEPs), mostly followed published patterns with CD105 expression as expected in a subset of MEPs. The immature CD38- HSCs, multipotent progenitors (MPPs), and lymphoid-primed MPPs (LMPPs) should be negative for the CD105 surface marker (Mori, et al, PNAS 2015) but we confirmed a very low expression (

Description: Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

Description: Early therapeutic decision-making is crucial in patients with higher-risk MDS where median survival is only around one year. Azacitidine prolongs survival for these patients (Fenaux et al, Lancet Oncology 2009) but clinically relevant biomarkers remain to be identified. We evaluated retrospectively, the impact of clinical parameters and mutational profiles in 134 consecutive patients treated with a median number of 7 cycles of Azacitidine (range 1-45), in accordance to European guidelines. The vast majority (n=114) had higher-risk disease i.e. MDS with IPSS int-2 or high, AML with multilinear dysplasia and 20-30% blasts or CMML-II. We combined an initial cohort from Karolinska University Hospital (n=89) with a validating cohort from King's College Hospital, London (n=45). Studied endpoints were response, as defined by the IWG criteria, and survival. Since prolonged survival is the main goal for this cohort of patients, we believe that survival is the most relevant endpoint, supported by the fact that even non-responding patients have a survival benefit from Azacitidine (Gore et al, Haematologica, 2013). While neither clinical parameters nor mutations had a significant impact on response rate, both karyotype and mutational profile were strongly associated with survival from the start of treatment, see Table 1 and Figure 1-2. IPSS high-risk cytogenetics was negatively associated with survival (median 20 vs 10 months; p

Description: Introduction: Azacytidine (Aza) is first-line treatment for patients with higher-risk MDS but only around 50% of patients respond to therapy. Overall survival for this patient group is short and clinical decision-making tools are highly warranted. As Aza may improve survival also in patients with hematologic improvement or stable disease, survival may be a better response predictor than response rate. Methods: We evaluated the impact of clinical parameters (n=134), mutations (n=90) and DNA methylation profiles (n=42) on response and survival in a cohort of consecutive patients with higher-risk MDS treated with Aza. Targeted sequencing of 42 genes involved in myeloid disease and Illumina 450 methylation arrays were applied for mutational assessment and methylation profiling, respectively. The IWG criteria were used for response scoring. Results: Patients were eligible for analysis if they had received ≥1 dose of Aza. Median number of cycles given was 6 (range 1-29). Responses were scored as CR (22%), mCR (11%), PR (3%), HI (13%), SD (27%) and PD (13%). Fifteen patients (11%) were not evaluated for response due to early death. Disease duration was negatively associated with both response (p=0.035) and survival (p=0.001). Adverse cytogenetics and high absolute neutrophil count was associated with shorter survival (p=0.03 and p=0.02) but not with response. No single mutation or group of mutations was associated with response although there was a weak positive trend for TET2 and ASXL1. When using survival as endpoint, ASXL1 showed a strong trend towards prolonged survival (median 29 vs 14 months, p=0.07) and, importantly, the group of patients with any mutation in histone modulators (ASXL1, EZH2, MLL) had a significant longer survival (median 28 vs 13 months, p=0.01). This remained significant in the cox regression model (HR 0.3223 (0.16-0.70 95% CI); p=0.002). No other mutations or group of mutations were associated with survival. Interestingly, previously reported negative prognostic factors including RUNX1 (p=0.82), TP53 (p=0.54), and the number of mutations (p=0.37), were not associated with survival in this Aza-treated cohort DNA methylation profiling identified 233 differentially methylated regions (DMRs) between responders and non-responders, corresponding to 200 genes, including six HOX-genes, which were highly enriched for gene ontology pathways involved in development and differentiation. High methylation of HOXA5, the most significant DMR, was associated with prolonged survival (22 vs 12 months, p=0.03). We also studied the methylation level of HOXA5 in CD34+ cells from patients with high-risk MDS and sorted compartments during myeloid differentiation in normal bone marrow. The methylation profile in responding patients was closer to that of differentiated cells while non-responding cells were closer to progenitor cells. Discussion: Single mutations have a limited impact on response rates. Howver, we demonstrate a clear survival benefit for patients with mutations in histone modulators, which previously have been reported as negative prognostic factors (Bejar, NEJM 2013; Haferlach, Leukemia 2014). Moreover, several negative risk factors, such as RUNX1, TP53, and the number of mutations were neutralized by Aza. Histone modulation mutations may therefore be used in the clinical decision-making for higher-risk MDS. We demonstrate for the first time that methylation profiles in genes involved in differentiation and development differ between responders and non-responders and that hypermethylation of HOXA5 is positively associated with survival (p=0.03). Since methylation pattern in HOXA5 is linked to differentiation status, we hypothesize that non-responding patients are skewed towards more immature differentiation. Figure 1: Survival curves Figure 1:. Survival curves Figure 2: DNA methylation levels at the HOXA5 locus. Squares represent gene location with light green=TSS-1500; Dark green=TSS-200; Red=Gene body; Magenta=1st Exon; Dark blue=5’UTR; Cyan=3’UTR and diamonds represent sample values. A=Median methylation level of responders illustrated with orange diamonds (MNCs) and non-responders with blue diamonds (MNCs). B=Added CD34+ cells with red diamonds. C=All patients. D=Normal bone marrow with PMN illustrated with brown diamonds and CMP with green diamonds. Figure 2:. DNA methylation levels at the HOXA5 locus. Squares represent gene location with light green=TSS-1500; Dark green=TSS-200; Red=Gene body; Magenta=1st Exon; Dark blue=5’UTR; Cyan=3’UTR and diamonds represent sample values. A=Median methylation level of responders illustrated with orange diamonds (MNCs) and non-responders with blue diamonds (MNCs). B=Added CD34+ cells with red diamonds. C=All patients. D=Normal bone marrow with PMN illustrated with brown diamonds and CMP with green diamonds. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Description: Purpose: Recurrently mutated genes have been identified in patients with myelodysplastic syndromes (MDS) and, more recently, in patients with unexplained cytopenia. (Kwok et al. Blood 2015, Hansen et al. American Journal of Hematology 2016 and Malcovati et al. Blood 2017). In this study, we investigated the prognostic impact of these mutated genes in patients with idiopathic cytopenia and compared them to a control cohort of patients with low risk MDS. Methods: We included patients with idiopathic cytopenia after routine assessment, without cytogenetic abnormalities. For comparison, a group of low risk MDS patients without cytogenetic abnormalities, excess of blasts or ring sideroblasts were included. All samples were sequenced covering at least the 20 most recurrently mutated genes in MDS, and a subset of cases underwent a blinded morphology review by two hematopathologists. Results: Two hundred and forty nine patients, 171 with idiopathic cytopenia and 78 with low risk MDS, were included in this study. Of these, 80 (47%) and 53 (68%), respectively, had one or more detectable mutations. There was no difference in survival between the groups, however a predefined subset of "adverse mutations" (ASXL1, NRAS, SRSF2, U2AF1, TP53, RUNX1, EZH2, IDH2 and GATA2, adopted from Bejar et al. Current Opinion in Hematology 2017) was associated with inferior survival in the MDS group (p= 0.035), but not in the group with idiopathic cytopenia and at least one mutation (p= 0.43) (Figure 1). However, if an adverse mutation was present in the idiopathic cytopenia group the risk of progression to MDS or AML increased significantly (HR [CI:95%] 12.01 [1.47; 98.23], p= 0.02), after adjusting for age and sex. Thus mutational screening identified the patients with unexplained cytopenia at risk of progressing to an overt myeloid neoplasm (Figure 2). A total of 18 patients (23%) progressed to a myeloid neoplasm during follow up, of those 12 had material available at time of progression. All patients who progressed to AML (n=4) acquired a new driver mutation at time of progression, in contrast to the patients who progressed to MDS or CMML (n=8) without excess of blasts, who showed a clonal expansion or a steady variant allele frequency at the time of progression. TET2 and DNMT3A mutations were more frequent in patients with idiopathic cytopenia, and were associated with less dysplasia of bone marrow cells. A total of 109 cases with idiopathic cytopenia underwent a blinded morphology review by two independent reviewers; ten cases were concordantly reclassified to fulfill the criteria for MDS, and all of these had at least two mutations. None of these have progressed to higher risk MDS and these ten are not included in the 18 patients mentioned above, who progressed to MDS, CMML or AML during follow up. Conclusion: We here show that mutational profiling can identify patients with idiopathic cytopenia who are at risk of progression, but in contrast to low-risk MDS, the presence of adverse mutations in patients with idiopathic cytopenia do not predict inferior survival. Disclosures Hansen: Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees. Grønbæk:Janssen Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees.

Description: Key Points SF3B1 mutations in MDS-RS have a multipotent lymphomyeloid origin. Transplantation of SF3B1 mutated MDS-RS HSCs into immune-deficient mice results in generation of characteristic ring sideroblasts.

Description: Deletions involving chromosome 7 constitute the second most common cytogenetic abnormality in myelodysplastic syndromes (MDS), associated with an overall poor outcome and high risk of transformation to acute myeloid leukemia (AML). Previous studies focusing on low to intermediate risk MDS, including cases with del(5q) as the only cytogenetic abnormality, have revealed a remarkably preserved stem and myeloid progenitor cell hierarchy, more characteristic of normal hematopoiesis than that of AML. Herein we explored this in a group of seven higher-risk MDS cases, all with an isolated deletion of the entire chromosome 7 (-7; monosomy 7), as the only cytogenetic abnormality. In all investigated isolated monosomy 7 cases, the expression of cell surface markers used to define the normal hematopoietic stem and myeloid progenitor cell hierarchy in bone marrow resembled more the expression pattern observed in AML than in low-risk MDS, irrespectively of the -7 MDS patients blast counts. Specifically, when compared to healthy age-matched controls, we observed a 15-fold reduction of LIN-CD34+CD38-CD45RA-CD90+ cells (hematopoietic stem cells; HSCs, p=0.010), a 15-fold increase in LIN-CD34+CD38+CD123+CD45RA+ cells (Granulocyte-macrophage progenitors; GMPs, p=0.038) and a 300-fold increase of LIN-CD34+CD38-CD90-CD45RA+ cells (lymphoid-primed multipotent progenitors; LMPPs, p=0.050) within total bone marrow mononuclear cells of MDS patients with isolated monosomy 7. Fluorescence in situ hybridization (FISH) analysis revealed high -7 clonal involvement (〉80%) for all investigated progenitor populations, whereas it was much lower (

Description: A high proportion of lower-risk del(5q) MDS patients will respond to treatment with lenalidomide. The estimated duration of transfusion-independence is 2 years including some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after start of treatment. As the molecular mechanisms underlying disease progression in del(5q) MDS remain to be elucidated, we do not know how to predict disease progression or how to monitor patients during lenalidomide treatment. We previously reported that small TP53 mutated subclones predict for an unfavorable outcome in del(5q) patients, and that these subclones expand with disease progression. However, whether or not other somatic mutations or factors related to the bone marrow microenvironment also contribute to disease progression has not been comprehensively assessed. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22), or other treatments including stem cell transplantation (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cells (HSPC) subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. In our cohort, 13 of 35 patients progressed to either higher-risk MDS (n=4) or leukemia (n=9), 12 of whom were treated with lenalidomide. Progression was associated with the detection of a restricted subset of new recurrent mutations, either alone or in combination: TP53 (n=9, p=0.0004), TET2 (n=6, p=0.006), RUNX1 (n=3, p=0.044), and PTPN11 (n=1). Regardless of whether the three mutations (TP53, TET2 and RUNX1) were present in the initial sample or whether they subsequently developed, testing positive for any of them carried a high probability (13/16, 81%) for predicting progression. For 11 out of 13 patients the new mutations were detected prior to the time point of clinical progression and the median time from detection of the mutation to clinical evidence of progression was 42 months (range 0-83.9). Thus, we were able to detect the mutation in the majority of cases well before clinical signs of disease progression. Seven of the nine patients who developed leukemia carried a TP53 mutation. Based on a median sequencing depth of 370 reads, the mutation was considered present pre-treatment in one of these patients and to have developed under treatment in the other six. Using flow cytometry for surveillance of HSPC subsets in lenalidomide-treated patients, we found that neither lenalidomide treatment nor the acquisition of additional mutations led to any uniform profound changes in the hematopoietic hierarchy unless the patient showed clinical signs of progression. Microarray analysis of mesenchymal stromal cells (MSC) exhibited an expression footprint consistent with MSC with high expression of typical MSC markers and absence of hematopoietic gene signatures. However, we observed only minor differences in gene expression between pre- treatment del(5q) and healthy MSC. In conclusion, while flow cytometric analysis of HSPC populations or analysis of the microenvironment had limited predictive value in this cohort of lower-risk del(5q) MDS, all patients who progressed to either higher-risk MDS or leukemia were identified by harboring recurrent mutations in a limited number of genes, i.e., TP53, RUNX1, TET2, and PTPN11. Based on our data, we advocate for conducting a prospective study aimed at investigating in a larger number of del(5q) MDS cases pre- and post-lenalidomide treatment, whether the detection of such mutations can guide clinical decision making, such as suggesting which patients should undergo hematopoietic cell transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Resistant Pseudomonas aeruginosa isolates are one of the major causes of both hospital-acquired infections (HAIs) and community-acquired infections (CAIs). However, management of P. aeruginosa infections is difficult as the bacterium is inherently resistant to many antibiotics. In this study, a collection of 75 P. aeruginosa clinical isolates from two tertiary hospitals from Athens and Alexnadroupolis in Greece was studied to assess antimicrobial sensitivity and molecular epidemiology. All P. aeruginosa isolates were tested for susceptibility to 11 commonly used antibiotics, and the newly introduced Double Locus Sequence Typing (DLST) scheme was implemented to elucidate the predominant clones. The tested P. aeruginosa isolates presented various resistant phenotypes, with Verona Integron-Mediated Metallo-β-lactamase (VIM-2) mechanisms being the majority, and a new phenotype, FEPR-CAZS, being reported for the first time in Greek isolates. DLST revealed two predominant types, 32-39 and 8-37, and provided evidence for intra-hospital transmission of the 32-39 clone in one of the hospitals. The results indicate that DLST can be a valuable tool when local outbreaks demand immediate tracking investigation with limited time and financial resources.

Description: The MDS subgroup refractory anemia with ring sideroblasts (RARS) is characterised by aberrant mitochondrial ferritin accumulation in erythroblasts that fail to mature into erythrocytes. Recently, dominant mutations in SF3B1, a core component of the spliceosome were demonstrated in 〉75% of RARS, but only in a minority of other MDS subtypes. Many RARS patients also carry other driver mutations, such as epigenetic mutations in DNMT3A and TET2, but the order of occurrence and cooperation between these mutations have not been established. We recently showed that SF3B1 suppresses the expression of the mitochondrial transporter protein ABCB7, which in turn mediates erythroid failure in RARS, but the link to clonal advantage of RARS hemopoietic stem cells (HSC) remains unclear. To explore this link, as well as the impact of additional mutations, we studied RARS with normal karyotype. Screening for 111 recurrently mutated genes in myeloid malignancies revealed SF3B1 in 12 out of 13 patients, TET2 mutations in 3 of these patients (Q916*, H1881Y, Q690*, and R1404*), and DNMT3A mutations in 3 patients(E240fs*8, F414L, W305*, E285*). Other mutations occurred only once. The frequencies of phenotypically defined RARS stem and myeloid-erythroid progenitor cells in the bone marrow (BM) did not differ from that of normal BM controls, whereas pro-B cells were significantly reduced in the RARS samples (p