Artigo Acesso aberto Revisado por pares

Proceedings of the CIMAGO Meeting – Challenges in Oncobiology Coimbra, 31st january 2020

2020; Wolters Kluwer; Volume: 99; Issue: 23 Linguagem: Inglês

10.1097/md.0000000000019821

ISSN

1536-5964

Tópico(s)

Phytochemical compounds biological activities

Resumo

Relevance of NF-kB and NRF2 transcription factors into multiple myeloma pathophysiology – A preliminary study Raquel Alves1,2,3, Ana Cristina Gonçalves1,2,3, Joana Jorge1,2,3, Catarina Afonso4, Adriana Roque4, Artur Paiva2,5, Letícia Ribeiro 2,4, Catarina Geraldes1,2,4, Ana Bela Sarmento-Ribeiro1,2,3,4 1Laboratory of Oncobiology and Hematology and University Clinic of Hematologly, Faculty of Medicine of University of Coimbra, Portugal;2iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal;5Flow Cytometry Operational Management Unit, Clinical Pathology Service, Centro Hospitalar e Universitário de Coimbra, Portugal. Multiple myeloma (MM) is a mature B cell dyscrasia characterized by an abnormal proliferation of monoclonal plasma cells, the presence of CRAB symptoms and/or myeloma-defining events. Bone marrow's microenvironment and nuclear factor-κB (NF-kB) signaling pathway play a relevant role in MM pathogenesis, constituting now therapeutic targets. Furthermore, oxidative stress has been observed in several hematopoietic malignancies, being the NF-kB and the nuclear factor (erythroid-derived 2)-like 2 (NRF2) the two key transcription factors (TFs) that regulate the cellular responses to oxidative stress and inflammation. This work aims to elucidate the importance of NF-kB and NRF2 axis in the MM biology and response to treatment. Thirty patients with MM were enrolled in the study and the bone marrow cell populations were evaluated by flow cytometry. The expression levels of NF-KB and its phosphorylated form, and of NRF2 were determined in the different cell populations, with a special focus on plasm cells, using flow cytometry. The TFs expression levels were correlated with clinical and laboratorial data. Our cohort of 30 MM patients included 12 females and 18 males, with a median age at diagnosis of 62 years (32–74). Two patients at diagnosis, fourteen patients in remission and fourteen in relapse. According with international scoring system (ISS) 12 patients (40%) were in stage I, 10 patients (33.3%) in stage II, 5 patients (16.7%) in stage III and the ISS was not determined in 3 patients (10%). In tumor plasma cells, we observed at diagnosis a higher percentage of cells expressing NRF2 (22.9 ± 22.7) and NF-kB (21.7 ± 21.5) comparing with observed at remission time-point (NRF2: 7.0 ± 3.1; NF-kB: 4.3 ± 1.5). An intermediated expression was observed (NRF2: 12.2 ± 4.3; NF-kB: 6.5 ± 1.8) in samples at relapse. The increase of tumor plasma cells expressing these TFs was also associated with the presence of bone lesion and kappa light-chains. All the other bone marrow populations expressed these TFs, but higher expression was observed in granulocytes. Our preliminary data suggest an association of NRF-2 and NF-kB expression with MM pathophysiology and disease stage. This work was supported by CIMAGO and NRC-LPCC/CIMAGO grant. STAT5 – A new therapeutic target in acute lymphoblastic leukemia? Joana Jorge1,2,3, Alexandre Estevam1, Raquel Alves1,2,3, Beatriz Lapa1,2,3, José Manuel Nascimento-Costa2,3,4, Ana Cristina Gonçalves1,2,3, Ana Bela Sarmento-Ribeiro1,2,3,5 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine of University of Coimbra (FMUC), Coimbra, Portugal;2iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4University Clinic of Oncology/Faculty of Medicine of University of Coimbra (FMUC), Coimbra, Portugal;5Clinical Hematology Department/Centro Hospital e Universitário de Coimbra, Coimbra, Portugal The signal transducer and activator of transcription (STAT) family members regulate the transcription of multiple genes involved in cell survival, proliferation, differentiation and apoptosis. Deregulation of STATs was associated with several hematological neoplasias such as acute lymphoblastic leukemia (ALL). ALL is a malignant transformation and proliferation of lymphoid progenitor cells in the bone marrow, blood and extramedullary sites. Several genetic and epigenetic alterations are involved in ALL pathogenesis that induce a cellular homeostasis unbalance resulting in differentiation arrest, high proliferation rates and/or resistance to apoptosis. Pimozide (PIM) is a STAT5 inhibitor that presented an anti-neoplastic effect in several other tumors. With this study, we assessed the therapeutic potential of PIM in in vitro models of ALL. To this end, two B-ALL cell lines (697 and REH) and two T-ALL (CEM and Jurkat) were incubated with PIM in single and daily administration schemes. Metabolic activity was evaluated by resazurin assay. Cell death was assessed by optic microscopy (May-Grünwald-Giemsa staining) and flow cytometry (FC), using the Annexin V and 7-AAD double staining. Cell cycle was evaluated by FC (IP/RNase). The expression levels of JAK/STAT target genes (CCNB1, CCNE1, CCND1, BCL2, KI-67, e HPRT1) were measured by qPCR. The statistical analysis was performed, considering a significance level of 95%. Our results showed that PIM reduced the metabolic activity in a time, dose and cell line dependent manner. T-ALL cell lines were more sensitive to PIM, with an IC50, at 72 h, of 7.5 μM (both CEM and Jurkat) comparing to the observed in B-ALL cells (10 μM for both, 697 and REH). Daily administration of 2.5 μM PIM demonstrated to be more effective than single administration of 7.5 μM. PIM induced cell death mainly by apoptosis, as observed by FC and by morphological analysis, and also cell cycle arrest in G0/G1 phase in all cell lines. Incubation with PIM also reduced the expression of JAK/STAT targeted genes. Our results suggest that PIM presents a therapeutic potential in in vitro ALL models and might be explored in other pre-clinical models. This work was supported by FMUC, CIMAGO and FCT (SFRH/BD/51994/2012). Aldehyde dehydrogenase polymorphisms: The role in myelodysplastic syndromes and acute myeloid leukemia Duarte Silva1, Ana Cristina Gonçalves1,2,3, Bárbara Marques1,4, Joana Jorge1,2,3, Raquel Alves1,2,3, André Barbosa Ribeiro2,4,Emília Cortesão1,2,3,4, José M. Nascimento Costa2,3,5, Ana Bela Sarmento-Ribeiro1,2,3,4 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra,Portugal;2iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4Clinical Hematology Department/Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal;5University Clinic of Oncology/Faculty of Medicine, University of Coimbra, Coimbra, Portugal Aldehyde dehydrogenase (ALDH) is highly expressed in hematopoietic stem cells (HSC) and inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Since myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) pathogenesis is associated with genetic and epigenetic changes in HSC, dysregulation of processes related with differentiation and cellular proliferation of these stem cells may be associated with the development of these hematological neoplasias. Thus, this study aims to analyze the role of ALDH1A2, ALDH3A1, and ALDH16A1 genetic variability in MDS and AML development and progression, in order to identify new potential risk factors and/or prognostic markers. ALDH single nucleotide polymorphisms (SNP) (ALDH1A2: rs4646626, ALDH3A1: rs2228100, ALDH3A1: rs887241 and ALDH16A1: rs1320303) were genotyped using tetra-primer-ARMS-PCR, in 99 MN patients (49 MDS patients and 50 AML patients), and in 118 control individuals. The role of SNPs in MDS and AML susceptibility and development/progression was assessed by logistic regression analysis. The overall survival was analyzed by Kaplan-Meier method. Our results show that ALDH3A1 (rs2228100) CG genotype was associated with MN and MDS development (MN: OR=2.072, 95%CI 1.197–3.585, p = 0.009; MDS: OR = 2.204, 95%CI 1.119–4.344, p = 0.022), while GG carriers have 3.4x lower risk of MN development (OR = 0.296, 95%CI 0.105–0.828, p = 0.02). Additionally, ALDH3A1 CG and GT haplotypes were associated with MDS risk (CG: OR = 1.901, 95%CI 1.181–3.062, p = 0.0104; GT: OR = 2.855, 95%CI 1.253–6.504, p = 0.0200), while CT haplotype carriers have 2.25x lower risk of MDS development (OR = 0.444, 95%CI 0.243–0.812, p = 0.0092). ALDH1A2 (rs4646626) GG genotype were associated with higher risk of MDS progression. In AML group, the ALDH1A2 (rs4646626) heterozygous carriers (AG) showed higher overall survival than homozygous carriers (AA + GG) (HR = 0.513, 95%CI 0.273–0.966, p = 0.035). Altogether, these findings reinforce the association of ALDH with MN susceptibility. The participation of ALDHs in oxidative stress and mutagenesis, contribute to the role of these enzymes in MN development, progression and prognosis. IREB2 rs17483548 single nucleotide polymorphism is associated with colorectal cancer Rui Gomes1, Ana Cristina Gonçalves1,2,3, Joana Jorge1,2,3, Raquel Alves1,2,3, Amélia Pereira1,4, Ana Bela Sarmento-Ribeiro1,2,3,5, José M. Nascimento Costa2,3,6 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology/Faculty of Medicine, University of Coimbra, Portugal;2iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4Medicine Department, Hospital Distrital da Figueira da Foz, EPE, Figueira da Foz, Portugal;5Clinical Hematology Department/Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal;6University Clinic of Oncology/Faculty of Medicine, University of Coimbra, Coimbra, Portugal Colorectal cancer (CRC) is one of the most common cancer worldwide. The CRC risk is determined by a complex interaction between environmental exposures and genetic variants. A growing body of evidence has proved that iron overload and dietary iron are associated with CRC. Considering the fundamental role of iron-regulatory protein 2 (IRP2) in the regulation of intracellular iron homeostasis, it is reasonable that some polymorphisms in IREB2 gene, which encode IRP2, could be involved in CRC carcinogenesis. In this context, we investigated the role of IREB2 rs17483548 single nucleotide polymorphism in CRC susceptibility, location, staging, and prognosis, in order to identify new potential risk factors and/or prognostic markers. To this end, a hospital-based case-control study with 83 CRC patients and 176 healthy controls was conducted. DNA from patients and controls was extracted from whole blood samples and the IREB2 rs17483548 corresponding genomic region was amplified by tetra-primer ARMS-PCR assay. The role of this variant in CRC susceptibility was performed by logistic regression analysis. The overall survival of patients stratified according to their genotypes was analyzed by Kaplan-Meier method (log-rank test and Cox proportional hazards model). The results showed that GA genotype (OR = 0.522, 95% CI 0.287–0.951, p = 0.034) and AA genotype (OR = 0.361, 95% CI 0.167–0.782, p = 0.010) were associated with a decreased susceptibility for CRC. On the other hand, GG genotype (OR = 2.133, 95% CI 1.212–3.755, p = 0.009) was found to increase the risk of CRC development. Additionally, GA and AA genotypes were associated with an increased predisposition for locoregional CRC. In contrast, GG genotype decreases the risk of right and transverse colon neoplasms. However, CRC patient's overall survival was not influenced by this genetic variant (HR = 1.269; 95% CI 0.615–2.620; p = 0.519). Altogether, these results suggest that IREB2 (rs17483548) single nucleotide polymorphism is associated with CRC development and may play an important role in tumor susceptibility and staging. However, more studies are needed to better characterize the impact of this single nucleotide polymorphism in CRC. NRF2 modulation as a new therapeutic approach in AML – In vitro studies Diana Figueiredo1, Joana Jorge1,2,3, Raquel Alves1,2,3, Beatriz Lapa1,2,3, Ana Cristina Gonçalves1,2,3, Ana Bela Sarmento-Ribeiro1,2,3,4 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Portugal;2iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal Oxidative stress (OS), characterized by an imbalance between the production of reactive oxygen species (ROS) and the biological system's ability to neutralize them, has been observed in most cancers, including acute myeloblastic leukemias (AML). An important mechanism in cellular defense against OS is the NRF2 pathway. This transcription factor protects normal cells from malignant transformation, regulating the expression of several antioxidant and detoxification genes. However, once malignant transformation occurs, NRF2 protects tumor cells from OS and preventing chemotherapy-induced cytotoxicity. Understanding the dual role of NRF2 is essential to consider it as a potential therapeutic target in AML. The aim of the present study was to analyze the effect of NRF2 activation and inhibition on AML cell lines. To achieve these goals, two AML cell lines (HEL and THP-1) were culture in absence and presence of ML385 (NRF2 inhibitor) and Oltipraz (NRF2 activator). The metabolic activity was evaluated by resazurin assay and cell death by optical microscopy (May-Grunwald staining) and flow cytometry (FC; Annexin V/7-AAD double staining). Cell cycle analysis was evaluated by FC, using a PI/RNAse solution. The intracellular levels of ROS and GSH were quantified by FC (DCFH2-DA, DHE and MO probes). The expression of NRF2-regulated genes (NFE2L2, KEAP1, NQO1, NFKB1) was tested by qPCR. The results were statistically analyzed considering a level of significance of 95% (p < 0.05). The results showed that both Oltipraz and ML385 decreased the metabolic activity of all cells, with ML385 showing a higher effect compared to Oltipraz (ML385 IC50 72 h: HEL = 30 μM, THP-1 = 65 μM; Oltipraz IC50 not reached). Both drugs induced a cytotoxic effect mediated by apoptosis. In THP-1 cells, a G0/G1 arrest were also observed. Additionally, cells incubated with both drugs showed an increase in the ROS/GSH ratio and in KEAP1 and NQO1 expression levels suggesting that Oltipraz may not be a NRF2 activator, at least in AML cells. The results suggest that AML cells are sensitive to NRF2 modulation and Oltipraz and ML385 may represent new potential therapeutic approaches in these hematological neoplasms. Epigenetics in chronic lymphocytic leukemia - Role in pathogenesis and treatment José Pedro Carda1,2,3,4, Raquel Alves1,2,3, Joana Jorge1,2,3, Sara Duarte4,Amélia Pereira2,5, Ilda P. Ribeiro2,3,6, Letícia Ribeiro2,4, Ana Cristina Gonçalves1,2, Isabel Marques Carreira2,5, Ana Bela Sarmento-Ribeiro1,2,3,4 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Portugal;2 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal;5Medicine Department, Hospital Distrital da Figueira da Foz, Figueira da Foz, Portugal;5Laboratory of Cytogenetics and Genomics /Faculty of Medicine, University of Coimbra, Coimbra, Portugal Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World. In recent years, more interest has been focused on the contribution of epigenetic changes to CLL development. In this context, the main goal of this work was to clarify the involvement of epigenetic modifications in CLL development, and correlate them with patients' clinical features, survival and prognostic risk groups as well as to analyze the therapeutic potential of epigenetic modulators. The methylation status of 25 tumor suppressor genes (ME001B, MRC-Holland) was carried out in 24 CLL patients with a median age of 70 years (range 50–81), 25% (n = 6) females and 75% (n = 18) males. Regarding Binet staging system, 19 (79%) were low risk (A and B) and five (21%) were high risk (C). Peripheral blood mononuclear cells (PBMCs) obtained from CLL patients were incubated in the absence and presence of decitabine (DAC), azacitidine (5-AC), vorinostat (SAHA) and/or panobinostat (PAN) in several concentrations, during 72 h. Cell viability will be evaluated by fluorometric microculture cytotoxicity assay (FMCA) every 24 h. Cell death were analyzed by flow cytometry (annexin V/7-AAD double stain). The results show that CLL patients have methylation in 13 of the 25 tumor suppressor genes studied, whereas controls only show methylation in the MSH6 gene [6/13 (46%)]. CLL patients have eight of these genes significantly hypermethylated [PAX5: 9/24 (38%); KLLN: 12/24 (50%); WT1: 22/24 (92%); CADM1: 9/24 (38%); THBS1: 13/24 (54%); CDH13: 15/24 (54%); TP53: 9/24 (38%); GATA5: 16/24 (67%)]. In addition, 58% (14/24) of CLL patients have 5 or more methylated genes and high-risk patients have a higher number of methylated genes (11/25) than low-risk patients (8/25). Epigenetic modulation revealed promising results in monotherapy. All drugs reduced the viability of CLL PBMCs (IC50: 30–40 μM 5-AC, 40–50 μM DAC, 50 nM PAN, 2.5–5 μM SAHA), inducing cell death by apoptosis with high specificity for neoplastic B cells. PAN and SAHA also induced a cytostatic effect. This study suggests that DNA methylation is a common event in CLL patients and that epigenetic modulators induce neoplastic B cells death. Therapeutic effect of Oltipraz in acute lymphoblastic leukemia – Preliminary in vitro studies Nisa Magalhães1,2, Diogo Roque1,2,; Maria Inês Costa2, Eduardo Gomes2, Joana Jorge2,3,4, Raquel Alves2,3,4, Beatriz Lapa2,3,4, Ana Cristina Gonçalves2,3,4, Ana Bela Sarmento-Ribeiro2,3,4,5 1Department of Chemistry, Faculty of Science and Technology, University of Coimbra;2Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;5Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal Acute lymphoblastic leukemia (ALL) is the most common hematological neoplasia in childhood, characterized by excessive proliferation of immature lymphoid cells and differentiation arrest, resulting in the accumulation of lymphoblasts and, consequently, the in suppression of normal hematopoiesis. In ALL there is an increase of reactive oxygen species (ROS) production and a decrease in antioxidant defenses, which makes evident the involvement of oxidative stress in the disease. The KEAP1-NRF2 pathway is an important mediator of the cytoprotective response to exogenous and endogenous stress induced by ROS. One of the major signaling proteins within this pathway is the transcription factor NRF2, which plays a vital role in cellular defense against oxidative stress through activation of various antioxidant genes. Thus, the aim of this study is to investigate the therapeutic potential of Oltipraz, an activator of NRF2, in T- and B-ALL cell lines. In this context, T-ALL (MOLT-4 and CEM) and B-ALL cell lines (697 and KOPN8) were cultured in the absence and presence of increasing concentrations of Oltipraz in monotherapy for 72 h. Metabolic activity was determined by the resazurin assay. Cell death was assessed with flow cytometry by double labeling with Annexin V and 7-AAD and through morphological analysis by Giemsa staining. Cell cycle was evaluated by flow cytometry using PI/RNase solution. The results were statistically analyzed considering a significance level of 95% (p < 0.05). Preliminary results showed that Oltipraz reduced metabolic activity in a time-, dose- and cell line dependent manner. After 72 h of treatment, B-ALL cells were more sensitive to Oltipraz (697: IC50 = 27.5 μM, KOPN8: IC50 = 4.5 μM) compared to T-ALL cells (MOLT-4: IC50 = 87.3 μM, CEM: IC50 = 40.1 μM). Oltipraz induced cell death by apoptosis in both B- and T-ALL subtypes (697 and MOLT-4). In summary, these preliminary results suggest that Oltipraz has an antiproliferative effect and that ALL-B cells are more sensitive to this drug. In addition, Oltipraz induces some cell death by apoptosis in LLA-T (MOLT-4) and LLA-B (697) cells. Zinc as a genoprotective player of DNA damage response – Preliminary results Maria Inês Costa1, Nisa Magalhães1,2; Diogo Roque1,2; Eduardo Gomes1, Raquel Alves1,3,4, Joana Jorge1,3,4, Beatriz Lapa1,3,4, Mafalda Laranjo3,4,5, Maria Filomena Botelho3,4,6, Ana Bela Sarmento-Ribeiro1,3,4,5, Ana Cristina Gonçalves1,3,4 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Portugal;2Department of Chemistry, Faculty of Science and Technology, University of Coimbra;3 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;4CACC-Clinical Academic Center of Coimbra, Portugal;5Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal;6Biophysics Institute/Faculty of Medicine, University of Coimbra, Portugal Micronutrients are important regulators of genomic stability, intervening in relevant pathways of the DNA damage response (DDR) as substrates and cofactors of numerous enzymes. Micronutrients' deficiency was already shown to cause similar effects as those produced by well-known genotoxic agents. Therefore, the adequate provision of micronutrients may be determinant for efficient DNA repair. Zinc is a vital micronutrient and a component of more than 300 enzymes involved in DDR. Increasing evidence has pointed its importance in DNA integrity and repair. The main aim of this study was to investigate the effects of zinc supplementation in cellular ability to handle genotoxic stimuli and DNA repair. The experiments were conducted on one human acute myeloid leukemia cell line (HEL) and on normal human lymphocytes (IMC), incubated for 2 days in RPMI-1640 medium with FBS (non-supplemented standard medium), zinc-depleted FBS and FBS plus 40 μM ZnSO4. Cells were then exposed to 10 μM H2O2 for 30 minutes. Chromosomal damage was analyzed using the cytokinesis-block micronucleus cytome assay. Two hours after H2O2 treatment, DNA repair was accessed with the alkaline comet assay. Our preliminary data shown that zinc supplementation resulted in a lower frequency of micronuclei and other chromosomal abnormalities in HEL and IMC (10 ± 1% and 7.8 ± 0.8%, respectively) when compared to non-supplemented conditions (14 ± 0% and 11.8 ± 0.3%). Zinc-depleted cells presented the highest frequency of chromosomal damage (20 ± 1% and 16.3 ± 3%), suggesting that zinc deficiency might affect cellular ability to cope with genotoxic stimuli. Nuclear buds were the most frequently found lesions in non-supplemented and zinc supplemented cells, whereas zinc depletion resulted mainly in micronuclei. Regarding the analysis of DNA repair, an initial comet assay on IMC shown a reduced DNA repair ability in cells cultured in zinc depletion and a slight recovery in cells from non-supplemented and supplemented mediums. These preliminary results suggest a protective role for zinc on chromosomal damage and a defective DDR when zinc is absent. EPO-EPOR axis as a prognostic and drug response biomarker in myelodysplastic syndrome – Preliminary results Beatriz Lapa1,2, Raquel S. Alves1,2,3, Joana Jorge1,2,3, Emília Cortesão1,2,3,4, Amélia Pereira2,5, Letícia Ribeiro2,3,4, Ana Cristina Gonçalves1,2,3, Ana B. Sarmento-Ribeiro1,2,3,4 1Laboratory of Oncobiology and Hematology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Portugal;2 iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;3CACC-Clinical Academic Center of Coimbra, Portugal;4Clinical Hematology Department, Centro Hospitalar Universitário de Coimbra;5Medicine Department, Hospital Distrital da Figueira da Foz, Portugal Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by peripheral blood cytopenias, ineffective hematopoiesis, dysplasia and tendency to acute myeloid leukemia transformation. Anemia is the more frequent cytopenia that could be treated erythropoietin (EPO) stimulating agents (ESA). EPO is the primary regulator of erythropoiesis and has pleiotropic effects. EPO analogs induce erythroid responses in few low-risk MDS patients and some develop neutralizing anti-EPO or anti-EPO receptor (EPOR) antibodies. The aim of this work was to asses EPO and EPOR expression in MDS patients in order to identify novel prognostic and drug response biomarkers. Six peripheral blood samples were collected from MDS patients, and stratified according to recombinant EPO treatment response, patients that respond to EPO treatment (n = 2) and patients that do not respond (n = 4). Flow cytometry was used to identify HSC and progenitor cells and assess the expression of EPO and EPOR. RNA from the samples was isolated and used to evaluate the expression levels of EPO and EPOR by qPCR. The non-responder patients presented a lower percentage of CD34+/CD117+ cells expressing EPOR (4.3 ± 1.4) as well as lower expression levels of EPOR (1810 ± 537) when compared do EPO responders (13.4 ± 1.8 and 4957 ± 414, respectively). The more differentiated progenitor cells (CD34+/CD45dim/CD117+/HLA-/CD13+/CD123+/CD105-) from non-responder patients presented a higher percentage of cells expressing EPOR (23.7 ± 4.8%) compared to responders (2.3 ± 0.5%) and expression levels like EPO responders (responders: 708 ± 65 MIF; non-responders: 772 ± 81 MIF). Furthermore, the responder patients presented an EPO gene expression 4-fold higher (0.50 ± 0.08) than the non-responders (0.13 ± 0.02) but EPOR gene expression presented a similar value (responders: 3.9 ± 0.3; non-responders: 4.3 ± 1.1). EPO responders and non-responders' MDS patients appeared to have different percentage of cells that express EPOR as well as different expression levels of EPO. Therefore, these preliminary results suggest that EPO/EPOR axis could be used as a predictive biomarker of therapy response. This project was funded by Liga Portuguesa Contra o Cancro and Sociedade Portugusa de Hematologia. The clinical value of Next-Generation Sequencing in Acute Myeloid Leukemia Bárbara Marques1,2, Adriana Roque1,2,3, Carolina Afonso1, Daniela Coelho1, João Gomes1, Ana Teresa Simões1, Margarida Coucelo1,2,3,4, Sandra Marini1, Letícia Ribeiro1,3,4, Ana Isabel Espadana1, Emília Cortesão1,2,3,4 1Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Portugal;2Faculty of Medicine of University of Coimbra, Portugal;3iCBR-Coimbra Institute for Clinical and Biomedical Research – Area of Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra and CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal;4CACC-Clinical Academic Center of Coimbra, Portugal; Contex: Next-Generation Sequencing (NGS), which analyze several genes at once, is useful in diseases with recurrent mutations. As Acute Myeloid Leukemia (AML) is highly heterogeneous, genetic profile study is worthwhile. Aim: to evaluate mutation pattern in AML with cytogenetic normal and not available karyotype. Methods: Characterization of mutation profiling by NGS in AML patients and correlation with outcome. Results: We analyzed 28 patients, 18 males, with median age 60(52–69) years. Karyotype was normal in 19 and not available in 9 patients. At least 1 driver mutation per patient was detected, median of 4 mutations (1–8), and 18 patients (64.3%) with ≥3 mutations. It was found mutations in NPM1[16], DNA methylation (DNMT3A[12], TET2[7], IDH1[4], IDH2[4]), cohesin complex (KMT2A[2], BCOR[4], STAG2[2], EZH2[1], SMC3[1]), splicing (SRSF2[1], SF3B1[1]), signaling pathway (NRAS[7], FLT3[10], KIT[1], PTPN11[5], CBL[1], JAK2[1]) and transcription factors (RUNX1[5], GATA1[1], GATA2[1

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