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Myeloproliferative neoplasms polycythemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis constitute a group of haematological diseases. The comprehensive assessment of signaling pathway activation in blood cells may aid the understanding of MPN pathophysiology. Thus, levels of post-translational protein modifications and total protein expression were determined in MPN patients and control leukocytes by using reverse-phase protein arrays (RPPA). Compared to control samples, p-SRC, p-CTNNB1, c-MYC, MCL-1, p-MDM2, BAX and CCNB1 showed higher expression in PV samples than controls. P-JAK2/JAK2 and pro-apoptotic BIM showed differential expression between JAK2V617F-positive and -negative ET patients. Apoptosis, cancer and PI3K/AKT pathways proteins showed differential expression among the studied groups. For most of the proteins analyzed using Western-Blot and RPPA, RPPA showed higher sensitivity to detect subtle differences. Taken together, our data indicate deregulated protein expression in MPN patients compared to controls. Thus, RPPA may be a useful method for broad proteome analysis in MPN patients´ leukocytes.

Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms.

Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil.

MicroRNAs are small non-coding RNAs that inhibit posttranscriptional gene expression through deadenylation and cleavage. Th ese molecules play crucial roles in regulating cell diff erentiation, proliferation and apoptosis [1]. MicroRNA expression levels are strongly associated with numerous human pathogenic diseases [2]. Th us, the discovery of microRNAs has opened up a wide range of possibilities regarding the understanding of disease pathophysiology and the development of new therapeutic agents. Recent studies show that microRNAs may participate in the regulation of apoptosis machinery (apoptomiRs). Apoptosis is a programmed cell death, orchestrated by the BCL-2 protein family, death receptors and inhibitor of apoptosis proteins (IAPs) [3]. Deregulated apoptosis is associated with tumorigenesis and autoimmunity [3] and seems to contribute to myeloproliferative neoplasms (MPNs), which are clonal disorders characterized by myeloaccumulation and in most cases by the presence of a somatic mutation in Janus kinase 2 (JAK2) [4]. Our research group previously described abnormal apoptosis in patients with MPNs [5 – 7]. We found abnormal expression of death-receptor pathway-related genes such as FAIM and C-FLIP in leukocytes and CD34 � hematopoietic stem cells (HSCs) [5], and deregulation of BCL-2 family members associated with JAK2 mutation [6,7]. Th ese fi ndings led us to investigate the molecular mechanisms associated with apoptosis deregulation in patients with MPNs. In this study, we sought to identify microRNAs whose targets are apoptosis-related genes, investigate their expression in patients with MPNs, and correlate this with the expression of apoptosis-related genes and JAK2 mutation allele burden using three algorithms (websites: http://www.diana.pcbi. upenn.edu/cgi-bin/miRGen/v3/Targets.cgi; Microcosm Targets: http://www.ebi.ac.uk/enright-srv/microcosm/cgi-bin/ targets/v5/hit_list.pl?genome_id � 2964; and Target Scan: http://www.targetscan.org). Th e microRNAs and their respective target genes (apoptosis-related genes) predicted by the bioinformatics tools are as follows: miR-15a: BIK and BCL-2; miR-16: BID, BIK, BCL-2 and BCL xl; miR-26a: C-FLIP, CIAP-2 and MCL-1; miR-130b: CIAP-2; miR-21: CIAP-2, FASL and BCL-2; miR-29c: CIAP-1 and MCL-1; and miR-let-7d: A1, BAX, FAS and FASL. In this study, we investigated apoptomiRs miR-26a, -130b, -21, -29c, -let-7d, -15a and -16. We quantifi ed their expression in bone marrow CD34 � HSCs and peripheral blood leukocytes in 27 patients with polycythemia vera (PV), 25 with essential thrombocythemia (ET) and 12 with primary myelofi brosis (PMF) (26 males and 38 females, mean age: 60.5 years). Th e bone marrow control group comprised 14 bone mar

Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation.

Background Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. Aims To evaluate expression of death receptors9 family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. Subjects and Methods Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2 −ΔΔCt . The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. Results In CD34 cells, there was mRNA overexpression for fas , faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas , c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. Conclusions The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV). This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005). CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively) in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020); while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19) compared with controls (1.39 and 2.01, p=0.006 and p=0.020). CD34+ cells in PV patients showed an elevated Bid expression (14.4) in comparison with healthy subjects (1.0; p=0.002). Patients' leukocytes showed an A1 augmentation (7.41, p=0.001) and a reduced expression of Bax (0.19; p=0.040) and Bad (0.2; p=0.030). There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV). Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005). Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente) em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020) e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19) em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0,020). Células CD34 + dos pacientes com PV mostraram expressão elevada de bid (14,4) em comparação aos controles (1,0; p = 0,002). Leucócitos dos pacientes mostraram aumento de A1 (7,41, p = 0,001) e expressão reduzida do Bax (0,19; p = 0,04) e Bad (0,2; p = 0,030). Não houve correlação entre percentagem de alelos JAK2 V617F mutados e expressão molecular. Pacientes com PV apresentaram alterações na expressão de moléculas Bcl-2 que podem interferir no controle da apoptose e contribuir para a patogênese da doença.

Abstract 5057 Background: Polycythaemia vera (PV) is a clonal disorder characterized by an accumulation of normal red and white cells, platelets, and their progenitors in absence of a definable stimulus. Despite the advances in PV diagnosis its physiopathology remains not well elucidated. Apoptosis deregulation seems to contribute to disease establishment. MicroRNAs are small noncoding RNAs, post transcriptional repressors of genes by deadenilation, which play relevant roles in regulation of apoptotic machinery. Here we investigate the relation of apoptosis deregulation and apoptomirs expression in PV patients. Aims: To quantify the apoptomirs −16, −21, −15a, −26a, 130b, 29c and let-7d expression in peripheral leukocytes and CD34+ hematopoietic stem cells (HSC) in Polycythemia Vera (PV) patients and to correlate with targets genes expression data. Subjects and Methods: 30 PV patients, 13 men and 17 women with a mean age (ma) of 64. 7y. 30 healthy subjects, 13 males and 17 females, ma=60. 2y and 23 bone marrow donors, 14 males and 9 females, ma=32. 6y. Peripheral leukocytes were obtained by Haes-Steril method and CD34+ hematopoietic stem cells were enriched by using the magnetically activated cell sorting, total RNA was extracted according to Trizol® method and High Capacity® Kit was used to synthesize cDNA. The microRNAs and apoptosis-related genes expression quantification was performed by real time PCR. Results were given as 2-ΔΔCt and statistical analyses were carried out by Mann-Whitney and Spearman tests. Western Blot was applied to detect Bcl-2 protein expression. Results: miR-16 and miR21 levels were increased in PV leukocytes (median= 3. 45 and 6. 97 respectively)) and CD34+ HSC (median= 3. 86 and 4. 07 respectively) in comparison to controls (control leukocytes median= 0. 483 and 0. 989 respectively) (CD34+ HSC median= 0. 978 and 1. 15, respectively) (p= 0. 0001 and 0. 0001 in leukocytes) and (p= 0. 0001 and 0. 0005 in CD34+ HSC, respectively). The Bcl-2 gene expression was decreased in PV leukocytes (m= 0.18) when compared to controls (m= 1. 03; p= 0. 019). In addition we found in CD34+ HSC negative correlation between miR-21 and anti-apoptotic gene Bcl-2 (r= −0. 417; p= 0. 033) and between miR16 and anti-apoptotic gene Bcl-2(r= −0. 384; p=0. 046). Bcl-2 western blot showed half expression in PV leukocytes when compared to controls. Conclusions: The results indicate the apoptomirs expression is linked to apoptosis deregulation in Polycythemia Vera physiopathology. Disclosures: No relevant conflicts of interest to declare.

Background Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34+ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34+ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34+ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-X L and BCLW. In contrast, pro-apoptotic BID and BIM EL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.