ALBERT

Description: Introduction: Risk stratification in acute myeloid leukemia (AML) is continually being refined as we learn more about the molecular pathophysiology of this heterogeneous disease. European LeukemiaNet (ELN) 2017 used widely to stratify the 'genetic' risk of AML, but there are considerable challenges in its application, particularly in centres where molecular genetic testing either not available, or not sufficiently timely for clinical decision making. Up to a third of the study subjects cannot be stratified using a full cytogenetic-molecular model in real-time, real-life setting. There are few attempts to combine clinical features and genetic factors aiming to find a scoring system to improve AML survival prediction. There is only one to our knowledge (Sorror ML et al. 2017). This study has generated an Adapted Genetic Risk (AGR) assessment, and used it in combination with clinical risk parameters to create a novel scoring system which has now been validated using two independent cohorts. Methods: A training cohort from São Paulo (FMUSP, n = 167) of intensively treated AML patients (18-65 years) was assessed using ELN2017 genetic criteria. A comparative validation with our AGR which permits missing cytogenetic or molecular data (Figure 1) split these patients into favorable-risk (FR), intermediate-risk (IR), and adverse-risk (AR). This cohort was also used for Cox Proportional-Hazard Model (CPHM) univariate and multivariate analysis to find clinical parameters that would inform a novel Survival AML Score (SAMLS). Variables which are included in SAMLS had to be either significant in both CPHM models or significant in univariate and crucial for multivariate fitness as measured for the Akaike Information Criterion (AIC). We then applied the AGR strategy and SAMLS to 2 independent test cohorts of intensively treated adult AML patients : Riberao Preto (FMRP, n=145) and Oxford (OUH, n=157). The study was approved by the institutional review boards of the 3 participating centers. Informed consent was obtained from all patients according to the Declaration of Helsinki. Results: Table 1 shows the clinical characteristics for all the 3 cohorts. The median follow-up (FUP) was 72.3, 44.4, and 70.5 months for FMUSP, FMRP, and OUH, respectively. The median Overall Survival (OS) was 12.4, 12.5, and 56.4 months and the 5-year OS were 29.6%, 29.7%, and 49.7% respectively. Both ELN2017 and AGR correlated with significant differences in OS (p-value

Description: Abstract 1520 Poster Board I-543 The adhesion of both red and white cells to the vessel walls of the microcirculation initiates vaso-occlusion in sickle cell disease (SCD). The chronic inflammatory nature of SCD leads to elevation of circulating cytokines in patients, which may contribute significantly to the activation of blood cells and their consequent adhesion. Nitric oxide (NO) has important inhibitory effects on cellular adhesive properties and drugs that enhance NO bioavailability or NO-cGMP-dependent signaling may hold potential for treatment of various aspects of SCD. This study aimed to observe the effect of cytokines, often found augmented in the plasma of SCD individuals, on the in vitro adhesive properties of neutrophils (neu) and red blood cells (RBC) from healthy control (CON) and steady-state SCD (SCD) individuals. Furthermore, the effects of BAY 73-6691, an inhibitor of the cGMP-hydrolyzing enzyme, phosphodiesterase 9A (PDE9A) and BAY 41-2271, a guanylate cylase activator, on non-stimulated and cytokine-stimulated cell adhesion were determined. Neus and RBCs were isolated from peripheral blood of CON and SCD individuals. Cell adhesion (5×106neu/ml in RPMI or 2×108RBC/ml in HBSS) to immobilized fibronectin (FN;20μg/ml) was assessed using static adhesion assays (30min, 37°C, 5%CO2) in the presence or absence of cytokines IL-8 (100-500ng/ml), TNF-alpha (0.1-1μg/ml) and GM-CSF (1-100ng/ml) and/or in the presence/absence of BAY 73-6691 (10-60μM), BAY 41-2271 (60-150nM) or DMSO vehicle (0.02%v/v). As previously demonstrated, SCDneu have a greater capacity to adhere to FN than CONneu (see Table). Stimulation of cells in vitro with all three cytokines significantly augmented both CONneu adhesion to FN and further increased SCDneu adhesion (Table). Co-incubation of both CONneu and SCDneu with BAY 73-6691, but not BAY 41-2271, significantly reduced their adhesions to FN (Table). Furthermore, BAY 73-6691, but essentially not BAY 41-2271, significantly inhibited CONneu and SCDneu adhesion stimulated by IL-8, TNF-alpha and GM-CSF (Table). DMSO vehicle had no significant effect upon neu adhesion (data not shown). As previously reported, SCD RBC have a greater capacity to adhere to FN, in vitro, compared to CON RBC (12.8±1.7% comp. 7.8±1.0%, n=7, p0.05) (1μg/ml TNF-alpha: CON RBC, 7.0±1.0%; SCD RBC, 11.4±1.9%, n=6, p〉0.05). Furthermore, BAY 73-6691 and BAY 41-2271 did not affect either basal CON RBC (data not shown) or SCD RBC adhesion (SCD RBC basal; 14.8±1.0%; 60μM BAY 73-6691, 16.4±1.3%; 150nM BAY 41-2271, 15.7±1.2%, n=6). Key SCD inflammatory mediators were found, at physiologically relevant concentrations, to augment the adhesive properties of neutrophils, but not RBC, from control and SCD individuals. Circulating inflammatory cytokines may play a role in the induction of leukocyte adhesive properties in SCD; in contrast factors other than inflammatory stimuli may be more important for induction of SCD RBC adhesion. Data suggest that elevation of intracellular cGMP may be an important approach for reducing SCD leukocyte, but not SCD RBC, adhesive properties, even in an inflammatory environment. PDE9A is highly expressed in hematopoietic cells and inhibition of this enzyme, with consequent augmentation of cGMP, may represent a tissue/cell-specific therapeutic drug target worthy of further in vitro and in vivo studies as a therapy for SCD. Table Effect of cytokine stimulation and cGMP-elevating agents upon adhesive properties of neutrophils from healthy control and steady-state SCD individuals % Neutrophil adhesion to Fibronectin (mean±SEM) Controls SCD Basal N=18 IL-8 (200ng/ml) N≥7 TNF-alpha (500ng/ml) N≥7 GM-CSF (100ng/ml) N≥7 Basal N=18 IL-8 (200ng/ml) N≥7 TNF-alpha (500ng/ml) N≥7 GM-CSF (100ng/ml) N≥7 Basal 16.1±1.5 26.6±2.8 27.4±5.5 21.4±1.6 21.4±1.9 32.5±3.5 29.2±3.0 25.2±2.2 * p

Description: Myelodysplastic syndromes (MDS) are a heterogeneous group of blood cancers characterized by bone marrow (BM) failure, peripheral blood cytopenias, dysplasia, chromosomal abnormalities and an increased risk for transformation to acute myeloid leukemia (AML). Patients (pts) with higher risk disease are primarily treated with pharmacologic treatments like hypomethylating therapy (HMT) (5-azacytidine and decitabine). 5-azacytidine (AZA) and decitabine (DAC) can result in overall response rates of 36% with a median duration of response of 15 months and 17-21% with a median duration of response of 10 months, respectively. Pts refractory to HMT have poor outcomes with a median overall survival of ∼4 months. Spliceosome gene mutations are frequently found in certain subtypes of MDS specifically SF3B1 (∼28%), U2AF1 (6-12%) and SRSF2 (6-12%). The prognostic value of spliceosome mutations in different MDS subtypes has been largely investigated while the impact of these mutations on treatment response is still unknown. We aim to investigate the frequency of three commonly mutated spliceosome genes (SF3B1, U2AF1, and SRSF2) in pts who failed HMT in order to define mutational frequency and evaluate the feasibility of targeted therapy with next generation spliceosome inhibitors. We screened a cohort of 120 pts (MDS, 70; MDS/MPN, 33; MDS/sAML, 17; median age: 69; male/female: 85/35) that underwent HMT (AZA: 58; DAC: 21; AZA/DAC: 7; AZA/REV: 25; DAC/REV: 4; AZA/DAC/REV: 5). Forty-eight percent of pts failed HMT therapy as refractory or relapse. We performed Sanger sequencing on BM/peripheral blood DNA for known pathways involved in MDS pathogenesis including methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), signaling (CBL, N/KRAS), transcription (RUNX1, TP53, JAK2), and RNA splicing (SF3B1, U2AF1, SRSF2). Data analysis was available for 90 pts. We detected a total of 131 mutations in different pathways. In total, spliceosome mutations were observed in 28/90 (31%) of pts. When we analyzed the presence of the mutations in relation to the rate of response, we found that pts who failed HMT have frequent spliceosome mutations: 17/58 (29%). We have reported that molecular mutations in TET2 and DNMT3A can predict response to treatment to HMT (Traina F, Blood (ASH Annual Meeting Abstracts), Nov 2011; 118: 461). Indeed, the frequency of mutations in methylation genes was lower in the group of pts who failed HMT (11/58; 18.9%) compared to pts who achieved hematological response (11/32; 34%). Spliceosome inhibitors have been proposed for targetted therapy in MDS. The presence of spliceosome mutations in pts who failed HMT can open a new era of investigation leading to the possibility of using spliceosome inhibitors in pts who fail conventional therapy. We performed RNA-sequencing analysis on BM cells of pts who failed HMT compared to pts who achieved hematological response (n=2 vs 2) in order to define any specific gene signature explaining the differences in response to HMT. We performed differential gene expression testing on 11,459 expressed genes. In total, 158 genes were differentially expressed at FDR 〈 .2 in responders compared to not responders. We identified several interesting genes involved in tumorigenesis and epigenetic regulation such as YPEL3, and ST14, which were up-regulated in responders vs not responders (FC: 4 and 7.5; P

Description: Abstract 461 Aberrant DNA methylation is a hallmark of myelodysplastic syndromes (MDS), MDS/myeloproliferative neoplasms (MDS/MPN) and secondary acute myeloid leukemia (sAML). It provides a rationale for treating these malignancies with hypomethylating agents like 5-azacitidine (AZA) and decitabine (DAC). However, treatment outcomes remain limited and heavily weighed on morphologic/cytogenetic results. The discovery of novel mutations has provided important insight into the pathogenesis of MDS and related disorders. Genes implicated in epigenetic regulation, including DNMT3A, TET2, IDH1/IDH2, EZH2, ASXL1 and UTX have been found mutated in MDS, while others have also been implicated in MDS pathogenesis. There is limited data on the predictive value of these genetic defects for treatment response and disease outcome. We hypothesized that these defects are important biomarkers predictive of response to hypomethylating agents. We studied 88 patients with MDS (RCUD=2, RARS=6, RCMD=11, MDS-U=3, RAEB-1/2=29, CMML1/2=16, MDS/MPN-U=5, RARS-T=5, AML from MDS=11) who received hypomethylating agents (AZA=53, DAC=24, both=11). The median number of cycles was 7 [range 1–35], median age was 69 years (range 42–82) and median follow-up was 18 months (range 0–76). Responses were scored according to IWG criteria. DNMT3A, TET2, IDH1/2, EZH2, ASXL1, UTX, KRAS, NRAS, CBL, RUNX1, TP53 and SF3B1 were sequenced using standard techniques. Categorical variables were analyzed using Chi-square statistics. Overall survival (OS) was analyzed using Kaplan-Meier; p-values ≤ 0.05 were considered statistically significant. Mutated patients were older than wild type (WT) cases (72 vs. 68 years, p=.01) but were well matched for marrow blast %, cytogenetic risk group and cycles of hypomethylating agents received. We found mutations in 40/88 (45%) patients. Mutations were most frequent in SF3B1 (6/11; 55%), ASXL1 (13/50; 26%), TET2 (18/88; 20%), KRAS (3/34; 9%), and DNMT3A (7/88; 8%). Less common were mutations in EZH2 (2/43; 5%), TP53 (1/23; 4%), IDH1 (4/88; 5%), IDH2 (3/88; 3%), and UTX (1/36;3%). No mutations were found in CBL, NRAS or RUNX1. Based on single mutations, overall response rate (ORR) was higher in mutated vs WT patients for DNMT3A (6/7 [86%] vs 33/81 [41%]; p=.02), ASXL1 (11/13 [85%] vs 14/37 [38%]; p=.003), and TET2 (12/18 [67%] vs. 27/70 [39%]; p=.03). All heterozygous DNMT3A mutants responded to hypomethylating agents. Differences remained significant when stratified to AZA treatment alone for DNMT3A (6/7 [86%] vs 21/56 [38%]; p=.01) and ASXL1 (9/11 [82%] vs 12/29 [41%]; p=.02) but not TET2 (6/10 [60%] vs 21/53 [40%]; p=0.22). The predictive value of combined mutations were analyzed for DNMT3A, TET2 and/or IDH1/2, showing better response to hypomethylating therapy in patients who had a mutation; ORR (mutated: 18/28 (64%) vs WT: 21/60 (35%); p=.01). This difference remained significant in patients receiving only AZA (n=53); ORR was 11/18 (61%) in mutant and 11/35 (31%) in WT patients (p=.03). No differences in ORR were noted for KRAS, EZH2 and IDH1/2 mutant and WT patients. No SF3B1 mutants responded to treatment while both patients with UTX and TP53 mutations responded. The frequency of AML evolution was also analyzed and showed no difference between mutant and WT cases for TET2 (7/18 [39%] vs 22/70 [31%];p=.52), ASXL1 (4/10 [40%] vs 11/35 [31%]; p=.61), and DNMT3A (3/7 [43%] vs 26/81 [32%];p=.56). No differences in OS and progression free survival (PFS) were noted between responders and non-responders to hypomethylating therapy (28 vs 17 mos, p=.25; 16 vs 8 mos, p=.54). Comparison of survival outcomes for mutant and WT patients showed no significant difference for DNMT3A (OS: 30 vs 21 mos, p=0.43; PFS: 20 vs 11, p=.53), ASXL1 (OS: 28 vs 22, p=.68; PFS: 16 vs 10, p=.88), and TET2 (OS: 30 vs 20 mos, p=.30). PFS was better in TET2 mutants compared to WT (19 vs 9, p=.03). No survival differences were noted between mutant and WT cases who responded to hypomethylating agents for DNMT3A (OS: 25 vs 28,p=.84; PFS: 14 vs 16, p=.78), ASXL1 (OS: 10 vs 18, p=.48; PFS: 10 vs 6, p=.76) TET2 (OS: 27 vs 16, p=.79; PFS: 18 vs10, p=.19). In conclusion, DNMT3A, ASXL1 and TET2 mutations were independently associated with a better response to hypomethylating drugs. Moreover, combined mutations in DNMT3A/TET2/IDH1/IDH2 may influence the response to hypomethylating agents, especially AZA supporting its role as a predictive biomarker in MDS treatment. Disclosures: Maciejewski: Celgene and Eisai, NIH, AA&MDS Foundation: Research Funding. Tiu:MDS Foundation Young Investigator Award: Research Funding.

Description: Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). GATA-1, a transcription factor essential for erythroid maturation was found to be upregulated in normal hematopoietic cells after erythroid differentiation, but not in differentiating MDS erythroblasts. ANKHD1 transcript variant 3, also named PP2500, (NM_024668), is a recently described gene that contains a GATA-1 binding site at its promoter region, and has been shown to be upregulated during erythroid differentiation of normal hematopoietic cells. We then sought to evaluate the expression of PP2500 in normal and MDS cells, the expression of GATA-1 and PP2500 in CD34+ normal and MDS cells during erythroid differentiation, as well as PP2500 functional consequences in terms of apoptosis. Bone marrow samples were obtained from 16 patients with a diagnosis of low-risk MDS (13 RA and 3 RARS) and 5 normal donors, and submitted to RNA extraction. For erythroid differentiation, bone marrow samples from one low-risk MDS patient and one normal donor were submitted to CD34+ selection using MIDI-MACS immunoaffinity columns and cells were plated on plastic culture dishes in methylcellulose medium with appropriate growth factors for 6 days. BFU-E, CFU-E and proerythroblasts were then cultured in alpha MEM for another 8 days. At days 6 and 14, cells were collected and submitted to RNA extraction and apoptosis analysis. Posttranscriptional PP2500 gene silencing was performed in Jurkat cells, through electroporation, using small interfering RNA (SMARTpool siRNA duplexes-Dharmacon). Forty-eight hours after transfection, cells were submitted to RNA extraction and apoptosis analysis. The expression level of mRNA was detected by real time RT-PCR. Endogenous controls used were GAPDH and b-actin. The relative quantification value of gene expression was calculated using 2−DDCT. Apoptosis was evaluated using annexin V-FITC/PI staining; erythroblast differentiation was verified with transferring-receptor/glycophorin-A and FACS analysis. Reduced expression of PP2500 was observed in low-risk MDS cells (RA/RARS) when compared with normal hematopoietic cells (mean ± SD; 0.15 ± 0.18 vs 1.07 ± 0.53, respectively; P=0.0004). Erythroid differentiation of CD34+ normal cells resulted in upregulation of PP2500 and GATA-1: 12-fold increase on day 14 compared to day 6, for both mRNAs. However, differentiating MDS erythroblasts failed to show a noteworthy increment in PP2500 and GATA-1 expressions: 1.5-fold increase for GATA-1 and 2-fold increase for PP2500, on day 14 compared to day 6. After erythroid differentiation, apoptotic cells accounted for 5.6% of cultured normal marrow samples on day 6 and 2.5% on day 14, whereas MDS cultures contained 4.8% apoptotic cells on day 6 and 9.5% on day 14. Posttranscriptional PP2500 gene silencing resulted in a 2-fold increase in apoptotic cells compared to control electroporated cells (27% vs 14% annexin V+/PI−, respectively). In conclusion, reduced expression of PP2500 mRNA was found in low-risk MDS cells compared with normal hematopoietic cells and MDS erythroblasts showed a defective GATA1 and PP2500 expression pattern during erythroid differentiation associated with an increased apoptosis. In addition, PP2500 gene silencing resulted in increasing apoptosis. These results suggest that PP2500 may have an anti-apoptotic function and a defective expression in MDS cells, which may be implicated in the pathophysiological process of MDS.

Description: Abstract 2419 Introduction: Acute leukemia and solid tumors result from alterations in the essential pathways of cell physiology including apoptosis, proliferation and genome instability. In solid tumors, the proapoptotic SIVA protein modulates apoptosis, proliferation, migration and promotes Stathmin inhibition through phosphorylation. Stathmin regulates microtubules dynamics and its hyperactivity confers chromosome instability in leukemia cells. Using two-hybrid system assay, we have identified SIVA as a binding partner of ANKHD1, an ankyrin-repeat-containing protein. ANKHD1 is overexpressed in acute myeloid leukemia (AML) and acute lymphoblast leukemia (ALL) and has the potential role of regulating multiple cellular functions via their repeat motifs. We thus hypothesized that ANKHD1 and SIVA could be involved in leukemogenesis. We aimed to evaluate SIVA expression in normal and leukemia hematopoietic cells, to confirm the endogenous ANKHD1/SIVA association, and to investigate the functional role of both proteins in apoptosis, proliferation, migration, and Stathmin activation. Materials and Methods: Expression of SIVA was evaluated by qPCR in total bone marrow cells from 22 healthy donors, 42 AML and 21 ALL patients at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. Leukemia cell lines (Jurkat, Namalwa or U937 cells) were used for functional studies. Endogenous protein interaction was verified by immunopreciptation and cofocal microscopy. We stably knocked down the endogenous expression level of ANKHD1 or SIVA with specific shRNA-expressing lentiviral vector and in vitro apoptosis was examined by AnnexinV/PI, cell growth by MTT assay and colony formation, and migration by transwell assays. In addition, we investigated in vivo tumor growth; leukemia cells were implanted in the dorsal sub cutis of NOD/SCID mice and tumors were excised, measured and weighed after 15 days. Stathmin activation proteins (Stathmin, phospho-Stathmin, alpha tubulin and acetylated-alpha tubulin) and apoptotic proteins (BCL-XL, BAX, JNK and phospho-JNK) were evaluated by Western blot. Appropriated statistical analysis was performed. Results: SIVA expression was significantly decreased in AML and ALL cells compared with normal hematopoietic cells (P