ALBERT

Description: Introduction: The detection of chromosomal abnormalities in myelodysplastic syndromes (MDS) supports the diagnosis, classification, prognostic stratification, therapy option, treatment monitoring and better understanding of the biology of disease. The main chromosomal abnormalities of MDS are losses and gains of genetic material, and these changes are among the most important prognostic parameters in IPSS-R. As a consequence, major advances have been achieved in the treatment and survival of patients. However, nearly half of the patients present a normal karyotype, which prevents their further characterization. Therefore, there is the desire to increase the abnormalities detection rate by other methods. Single nucleotide polymorphisms array (SNPa), also referred to as chromosomal microarray, is a sensitive technology used to perform high-resolution genome-wide DNA losses, gains and copy-neutral loss of heterozygosity (CN-LOH). The genomic DNA is hybridized to polymorphic probes which are SNP markers and non-polymorphic probes which are copy number markers, providing information about CN-LOH and copy number alteration (CNA), respectively. Previous studies have shown that the addition of the SNPa to karyotype (KT) increases by 28% the detection rate of cytogenetic abnormalities. Objective: To substantiate this idea we describe two cases of MDS to whom SNPa added valued information to karyotype. Methods: DNA was extracted from bone marrow cells for genomic clonal evaluation. The DNA was digested, amplified, fragmented, labeled and hybridized to the chip (Affymetrix - CytoScan HD®) containing the probes. The chip was scanned to detect the signals' intensity emitted by the hybridized probes which were further analyzed by a software (ChAS) that allows visualization of CNA and CN-LOH. CNA´s analysis relies on the comparison of the obtained signals to a reference diploid DNA signal, and the difference encountered is characterized as loss or gain. CN-LOH analysis is based on two possible nucleotide signals (A or B), which are evaluated to discriminate three genotypes: AA, AB and BB. CN-LOH occurs when one allele is lost and duplicate another, resulting in genotypes AA or BB. Results: Case 1, 64y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II fibrosis. KT: 46,XY,del(5)(q15q33),del(17)(p11.2)[16] / 46,XY[4]. SNPa: 3p21.31p21.2 CN-LOH; 5q21.1q35.3 loss (CN:1.00); -7; +8; 12p13.33p12.3, 12p12.1p11.22, 12q22q23.3 loss (CN:1.00); -16; 17p13.3p11.2 mosaic loss (CN: 1.50) and -Y. Case 2, 74y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II/lll fibrosis. KT: 46,XY[15]. SNPa: 21q21.1q22.3 CN-LOH. Discussion and Conclusion: The SNPa has the advantage of detecting genomic alterations regardless of the cell cycle, even when the cell is quiescent or growth is defective. It also enables the identification of CN-LOH (also known as uniparental disomy, UPD), submicroscopic amplifications and deletions that are not detected by KT. On the other hand, SNPa does not allow the identification of balanced translocations and polyploidy. In case 1, after SNPa analysis, some chromosomal abnormalities (−7, +8, −16 and −Y) were found in sporadic metaphases during the KT reanalysis, but had not initially been described because they did not meet the criterion to be considered as a cytogenetic clone. The risk-stratification (IPSS-R) for this patient was intermediate, but the addition of the SNPa results the risk-stratification could be changed to very poor. Seven months after diagnosis the patient developed acute myeloid leukemia and died. In case 2, CN-LOH detected by SNPa could be responsible for homozygosity of mutations in critical genes located in the 21p region, such as RUNX1 that encodes a protein, which is a transcription factor critical in hematopoiesis. Indeed, sequencing of candidate genes in CN-LOH regions should be considered a priority in the search of driver mutations of MDS. Twenty-four months after diagnosis the patient died due to other non-hematologic causes. In Summary, SNPa analysis may add value to KT non-informative results and occasionally reveal cryptic abnormalities not recognized by karyotyping. However, SNPa analysis should be viewed as a complimentary tool. Acknowledgment: The SNPa test was supported by Grupo Fleury Disclosures No relevant conflicts of interest to declare.

Description: Thrombotic events are the main complication of Philadelphia-negative chronic myeloproliferative neoplasms (MPN). In polycythemia vera (PV) and essential thrombocythemia (ET), risk factors for thrombosis are well established, such as age greater than 60 years and previous thrombosis. However, the role of JAK2 V617F mutation and leukocytosis at diagnosis as risk factor for thrombosis is still controversial. Our aim was to identify factors related to the risk for thrombotic events in the studied population.This study is a retrospective non-interventional cohort. All of the analyses were performed using the database of 142 patients with MPN regularly followed at the Hematology Division (at UNIFESP-SP) from 1992 to 2014. Diagnosis was established according to WHO criteria. We analyzed the JAK2 V617F mutation, hemoglobin (g/dL), hematocrit (%), white blood cells (x109/L) and platelets (x109/L) at the diagnosis and DIPSS-Plus risk score (International Working Group for Myelofibrosis Research and Treatment, 2009). These variables were associated with thrombotic event at any time.Of the 142 patients, 54 had diagnosis of PMF, 28 of PV, 33 of ET and 27 of post-essential thrombocythaemic myelofibrosis (post ET MF) or post-polycythaemic myelofibrosis (post-PV MF). This last group was included in myelofibrosis group for statistical purposes. Thrombotic events were more frequent in PV patients (39.2%), followed by ET (33.3%), and PMF (20.9%). From those which JAK2 mutation was obtained, it was positive in 92.4% of PV patients, 62% of PMF and 50% of ET. In none of the three groups, the presence of JAK2 V617F mutation was related to increased risk of thrombosis. In myelofibrosis, leukocytosis was higher among thrombotic patients (median of 13.7 in thrombotic group versus 9.7x 109/L; p 0.0379). None of the other parameters, hemoglobin, hematocrit, platelets and DIPSS-Plus were statistically significant. In ET, the hemoglobin level at diagnosis was significantly higher in the presence of thrombosis (mean of 14.57 in thrombotic group against 13.03 g/dL in the non-thrombotic one, p 0.0428). The other parameters, hematocrit, white blood cells and platelets were not relevant. The median WBC in the thrombotic group was 9.4 and in the non-thrombotic one 9.3 x109/L. Finally, in polycythemia vera, none of the variables were related to thrombosis. Among the studied population, leukocytosis was increased in patients with thrombotic event in MF. Thus, monitoring leukocyte count in MF is essential to predict thrombosis risk and should be further studied in order to define therapeutic goals in these patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Lipids are molecules that stand out among the different cellular metabolites by their enormous molecular diversity. Their functions were initially related to the composition of biological membranes and energy storage, but currently, these molecules have been analyzed considering different functions and regulatory signaling (Loizides-Mangold, 2013). It is known that lipid membranes and lipid mediators constitute specific phenotypes, including tumors (Hilvo et al, 2011). Lipid metabolism in cancer had been studied predominantly at the genetic level has recently gained further interest. Lipidomics studies show a powerful means of investigating pathophysiological issues and involvement of lipids in pathological states for both diseases in which lipids are known to play a role, but also for those which role is not well characterized (Roberts et al., 2008). Myeloid neoplasms are clonal diseases of the hematopoietic stem cell which can be present in the bone marrow and/or peripheral blood. Over the past two decades, mass spectrometry (MS) has emerged as the main method used in lipidomics analysis, which allows the structural characterization and quantification of complex lipids and their metabolites (MURPHY et al, 2005). Due to the importance of this field we have considered the use of the lipidomic innovative platform to identify differences in the plasma lipid metabolomic profile of hematological patients with Myeloid Neoplasms. Methods: Untargeted Shotgun MS/MS Analysis was performed on an independent service at the AB-Sciex Laboratory located in Sao Paulo, SP, Brazil on a 5600 Triple TOF mass spectrometer (ABSciex) instrument with an acquisition scan rate of 100 spectra/sec and stable mass accuracy of ~2 ppm. Plasma samples from 153 participants were analyzed being, 90 of the Control Group, 43 Myeloproliferative Neoplasms (MPN), 11 Myelodysplastic Syndromes (MDS) and 9 Acute Myeloid Leukemias (AML). Data were acquired using the AB-Sciex Analyst TF, processed using the AB-Sciex LipidViewTM and the web-based analytical pipeline MetaboAnalyst 2.0 (www.metaboanalyst.ca) (Xia et al, 2012). Results: Untargeted analysis identified in negative and positive-modes a total of 658 features at 2 ppm resolution. PCA and PLS-DA analysis revealed clear discrimination among groups, in particular for AML patients. Main lipid groups differentially expressed were: Monoacylglycerols (MAG), Glucosylceramide E (GlcdE), Ethyl Esters (EE), Lysophosphatidic acid (LPA), Sulfoquinovosil diacylglycerols (SQDG), Monoglycerols (MG), Methyl Ethanolamines (ME), Lysophosphatidylcholines (LPC), Dimethyl Phosfatidyletanilamines (DMPE), Monometylphosphatidiletanolamines (MMPE), Ceramide-1-phosphate (CerP), Glicerophosphoglycerols (GP), Lysomonomethyl Glycerophosphocholine (LMMPE), Phosphatidic Acids (PA), Ergosterols (ERG), Glycerophosphoserine (PS), Diacylglycerols (DAG), Hexocylceramides (HexCer) and Lanosterol (Lan). ROC Curve Analysis revealed Total LMMPE as the strongest discriminating marker between Controls from Patients with MDS or AML (Sensitivity= 0.95 (0.824-1); Specificity= 0.8941 (0.847-0953); Positive Likelihood Ratio= 8.972 and Negative Likelihood Ratio =0.05592 and T Test= 7.576E-12). In addition these lipids were also able to differentiate MDS and AML from NMP (Sensitivity= 0.9118 (0.824-1), Specificity= 0.95 (0.85-1), Positive Likelihood Ratio= 18.2 and Negative Likelihood Ratio= 0.05592). Conclusions: The Myeloproliferative Neoplasms from the point of view of global plasma lipidomics are accompanied by several modifications. In particular the Lysomonomethyl-Phosphatidylethanolamines (LMMPE) seems to play important differentiating roles among them. Disclosures No relevant conflicts of interest to declare.

Description: Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. Objectives: We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Material and Methods: Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. Results: We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2, RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74, 96, and 21% of the MM samples, respectively. Conclusions: Analysis of differential expression using SAGE could identify new genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

Description: Abstract 3536 ΔNp73 is an alternative TP73 gene transcript lacking the transactivation (TA) domain that is generated via alternative splicing and/or P2 promoter. The encoded protein acts as a potent transdominant inhibitor of wild type TP53 and full-length TAp73. In several human malignancies the unbalanced expression of transcriptionally active (TAp73) and inactive (ΔNp73) variants correlates with treatment outcome. We have previously reported that higher ΔN/TA isoform expression ratio was associated with poorer prognosis and resistance to cytarabine induced apoptosis in patients with acute myeloid leukemia (AML) (Lucena-Araujo et al., 2008). In acute promyelocytic leukemia (APL), both isoforms are expressed, but the clinical significance remains unknown. The aim of this study was to determine whether the ΔN/TA expression ratio was associated with treatment outcome of APL patients and to investigate the mechanisms by which ΔNp73 may contribute to PML-RARa+ cell survival. Using isoform-specific probes for ΔNp73 and TAp73, their expression was analyzed in 166 APL patients by Real-time quantitative polymerase chain reaction (RQ-PCR). Patients were divided into tertiles for ΔN/TA expression ratio (median value=23.62; 33rd/66th percentiles=12.8/42.3) and their clinical and laboratory characteristics were compared. Patients in the highest tertile presented higher white blood cells (WBC) counts than those in intermediate/lower tertiles (p

Description: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by t(15;17)(q22;q21), a balanced reciprocal translocation involving PML and RARA genes. Studies conducted in animal models with PML/RARA suggest that this gene rearrangement is not sufficient for the development of APL, emphasizing the importance of additional mutations. Another gene that could be involved in APL leukemogenesis is FLT3, a Fms-like tyrosine kinase receptor which mutations have been observed in different AML subtypes as the internal tandem duplications (ITD), in around 18–37% of APL cases or less frequently, the D835Y mutation. The association of FLT3/ITD in APL gave rise to the proposition that it could provide a common class of cooperative mutation in the development of the disease. Indeed, APL represents 21% of all cases of AML with karyotype confirmation in our institution, located in São Paulo, Southeast region of Brazil. Bone marrow samples from 92 consecutive APL patients at diagnosis were enrolled in the study. Mean age was 29.5 years old, varying from 4 months to 67 years old, with the following age distribution: 35.7% of the patients were younger than 20 years old; 55.2% were between 20 and 49 years old and only 17.2% were older than 50 years. There were 50 males and 42 females. The WBC count demonstrated a wide variation, from severe leukopenia (