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O presente estudo tem como objetivo avaliar se a descompressão simples da cabeça femoral nos estágios iniciais da osteonecrose da cabeça femoral melhora a percepção subjetiva da dor dos pacientes e se evita a progressão da doença para o colapso da cabeça femoral e a indicação final de artroplastia total do quadril.

Investigar o uso de drenos em uma população controlada de pacientes com osteoartrose do quadril submetidos a artroplastia total de quadril primária.

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by ineffective hematopoiesis and risk of leukemia transformation. There is evidence to suggest the participation of immune system deregulation in MDS pathogenesis. Interleukin-32 (IL-32) is a newly described multifunctional cytokine reported as an important mediator in autoimmune and inflammatory disorders. In the present study, we reported the expression of IL32and IL32transcript variants (α, β, γand δ) in peripheral blood CD3+cells from healthy controls and MDS patients. CD3+cells were isolated by immunomagnetic cell sorting from thirty-nine untreated MDS patients and twenty-nine healthy donors. Gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Kruskal-Wallis test with Dunns post test and Log-rank (Mantel-Cox) were used, as appropriate. A pvalue <0.05 was considered statistically significant. IL32expression and IL32transcript variants IL32α, IL32β, IL32γ,and IL32δ, were similar in peripheral blood CD3+cells from healthy donors and MDS patients. Increased IL-32α expression was an independent predictor for MDS disease progression by univariate and multivariate analysis. We observed that IL32expression is not differently expressed in CD3+cells from MDS patients; nevertheless IL32αhas a potential role in disease progression.

SIVAis a transcriptional target of p53 that plays a potential role in the development and progression of cancer. In this study, we analyzed SIVA1and SIVA2expression, and its association with clinical features and TP53and MDM2expression in bone marrow cells from healthy donors and myelodysplastic syndrome (MDS) patients. Fifty-five untreated patients with MDS and 22 healthy donors were included. Gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Spearman correlation analysis and Log-rank (Mantel-Cox) were used, as appropriate. A pvalue <0.05 was considered statistically significant. SIVA1and SIVA2transcripts were significantly decreased in bone marrow samples from MDS patients compared to healthy donors, and positively correlated with MDM2and TP53expression in MDS patients (all p< 0.05). MDM2expression was also downregulated in bone marrow samples from MDS patients compared to healthy donors (p< 0.05). However, SIVA1, SIVA2, MDM2and TP53expressions did not impact on MDS outcomes. SIVA1and SIVA2transcripts are downregulated in bone marrow samples from MDS patients.

Primary myelofibrosis is a Philadelphia-negative myeloproliferative neoplasm characterized by clonal myeloid expansion, followed by progressive fibrous connective tissue deposition in the bone marrow, resulting in bone marrow failure. Clonal evolution can also occur, with an increased risk of transformation to acute myeloid leukemia. In addition, disabling constitutional symptoms secondary to the high circulating levels of proinflammatory cytokines and hepatosplenomegaly frequently impair quality of life. Herein the main current treatment options for primary myelofibrosis patients are discussed, contemplating disease-modifying therapeutics in addition to palliative measures, in an individualized patient-based approach.

Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia.

Although myelodysplastic syndromes have a clear definition in theory, the morphologic dysplasia associated with ineffective hematopoiesis may be subtle and difficult to recognize and can commonly be mimicked by systemic conditions, such as infections, autoimmune disorders, nutritional deficiencies, toxic factors and non-hematological malignancies. However, myelodysplastic syndromes may truly coexist with other systemic diseases, which can be masked when the patient's symptoms are attributed exclusively to myelodysplastic syndromes without further investigation. To better illustrate this, we herein describe two cases associated with synchronous gastric cancers.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Background:Insulin receptor substrate 2 (IRS2) mediates mitogenic and antiapoptotic signaling from IR, IGF1R, EPOR and TPOR. Studies performed in non-hematological lineages have shown that IRS2 associates with proteins involved in the JAK/STAT, PI3K/Akt/mTOR, and Ras/Raf/MAPK pathways, known to be involved in cell proliferation and survival. Primary Myelofibrosis (PMF), Polycythemia Vera (PV), and Essential Thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPN) frequently associated with the JAK2V617F mutation, and with hyperactivation of the JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAPK pathways. Ruxolitinib was designed to target JAK2, but the lack of complete clinical response in most patients indicates the need for identifying other signaling pathways that may cooperate with JAK2. We have previously reported that IRS2 constitutively associates with JAK2 in HEL cells (JAK2V617F), though not in U937 and NB4 lineages (JAK2wild type). IRS2mRNA expression in CD34+cells was significantly higher in ET patients compared to healthy donors, and in patients harboring the JAK2V617F mutation versusJAK2wild type, which suggests that IRS2 is involved in cell proliferation in MPN. We herein aim to characterize the effects of IRS2silencing and to evaluate the synergistic effect of IRS2 silencing and ruxolitinib treatment on cell growth, clonogenicity, apoptosis, and on the JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAPK signaling pathways in JAK2V617F cells treated or not with ruxolitinib.

Mast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug.

Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are BCR-ABL1 negative Chronic Myeloproliferative Neoplasms (MPN) characterized by increased myeloid proliferation, with predominant erythroid, megakaryocytic and megakaryocytic/granulocytic expansion, respectively. The finding of a recurrent mutation in the gene of the tyrosine-kinase Janus kinase 2 (JAK2 V617F) in these diseases has raised the hypothesis that this could be the main cause of their development. However, the evidence that MPN patients have a very similar response to JAK2 inhibitors regardless of JAK2 mutation status, and the knowledge that many receptors and substrates may lead to the activation of JAK/STAT, Ras/Raf/MAP kinases and PI3K/Akt/mTOR pathways, indicate the need to investigate other crucial proteins involved in the physiopathology of these diseases. Insulin receptor substrate 2 (IRS2) mediates mitogenic and antiapoptotic signaling from IR, IGF-IR, EPO-R and TPO-R. Previous studies performed on non-hematological cell lines have shown the association of IRS2 with JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAP kinases pathways, giving rise to the hypothesis that IRS2 could participate in the activation of crucial signaling pathways in MPN through direct interaction with JAK2 or through alternative mechanisms.To identify the JAK2/IRS2 protein interaction and to study the effects of pharmacological JAK1/2 inhibition (Ruxolitinib) over IRS2 phosphorylation in leukemia cell lines harboring or not the JAK2 V617F mutation; to characterize IRS2 expression in CD34+ cells from patients with MPN and its correlation with clinical data including JAK2 mutation status.Leukemia cell lines carrying JAK2 V617F mutation (HEL) or not (HL60) were used for immunoprecipitation and immunobloting with IRS2 and JAK2 antibodies. Cells treated or not with JAK1/2 inhibitor Ruxolitinib were also submitted to immunoprecipitation and immunobloting with IRS2 and anti-phosphotyrosine antibodies. Peripheral blood mononuclear cells from 28 healthy donors and 97 patients with MPN (PV=28, ET=38, PMF=31) were included, and CD34+ cells were submitted to quantitative PCR (q-PCR). Relative expression of IRS2 was correlated with clinical data and with JAK2 V617F mutation status.Immunoprecipitation analysis showed that IRS2 associates with JAK2 in leukemia cell lines harboring (HEL) or not (HL60) the JAK2 V617F mutation. Furthermore, treatment of HEL cell line with the JAK1/2 selective inhibitor Ruxolitinib resulted in decreased IRS2 tyrosine phosphorylation. IRS2 mRNA expression in CD34+ cells were significantly higher in patients with ET when compared to healthy donors (1.70 [0.42-10.60] versus 0.87 [0.01-11.22], p=0.03). There was no difference in IRS2 mRNA expression in PV or PMF patients when compared to healthy donors. Furthermore, significantly higher levels of IRS2 mRNA expression were observed in patients harboring JAK2 V617F mutation when compared to the wild type JAK2 for ET (2.37 [0.96-10.60], n=14 versus 1.54 [0.42-1.54], n=22; p=0.01); and for PMF (2.27 [0.003-10.59], n=20 versus 0.60 [0.02-2.42], n=11; p=0.02). Although there was also a significant difference in IRS2 mRNA expression in mutated versus non mutated JAK2 in PV (p=0.02), the number of non mutated samples was low (n=2).Our data indicate that IRS2 is a binding partner of JAK2 in myeloproliferative neoplasms and suggest that this protein association may be involved in cell proliferation in these diseases. The higher IRS2 expression in mutated samples (JAK2 V617F) might be associated with the constitutive activation of JAK2 in these samples.No relevant conflicts of interest to declare.

Mast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug.To characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors.Four individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter]), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture.Case 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen.We herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013.No relevant conflicts of interest to declare.

Acquired mutations in TET2 and DNMT3A have been found in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and may predict a worse survival in these diseases. TET2 mutations are considered to be a loss-of-function mutation and results in decreased 5-hydroxymethylcitosine (5-hmc) levels. In normal CD34+ cells, TET2 silencing skews progenitor differentiation towards the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. Dnmt3a participates in the epigenetic silencing of hematopoietic stem cell regulatory genes, enabling efficient differentiation. Here, we attempted to evaluate the expression of TET2 and DNMT3A in total bone marrow cells from normal donors, patients with MDS and AML, and in CD34+ cells from MDS and normal controls during erythroid differentiation.The study included normal donors (n = 21), patients with MDS (n = 43) and AML (n = 42) at diagnosis. All normal donors and patients provided informed written consent and the study was approved by the ethics committee of the Institution. MDS patients were stratified into low and high-risk according to WHO classification (RCUD/RCMD/RARS=31 and RAEB1/RAEB2=12). TET2 and DNMT3A mRNA expression was assessed by quantitative PCR. CD34+ cells from normal donors (n = 9) and low-risk MDS patients (n = 7) were submitted to erythroid differentiation. Cells were collected and submitted to immunophenotyping for GPA and CD71 (days 6 and 12) and q-PCR for TET2 and DNMT3A expression (days 6, 8 and 12). Results of gene expression in normal donors and patients are presented as median, minimum-maximum, and were compared using Mann-Whitney test. Student t test was used for comparison of gene expression during CD34+ erythroid diferentiation. Overall survival was defined from the time of sampling to the date of death or last seen. Univariate analysis for overall survival was conducted with the Cox proportional hazards model.TET2 expression was significantly reduced in both AML (0.62; 0.01–32.69) and MDS (1.46; 0.17–21.30) compared to normal donors (2.72; 0.43–31.49); P<0.0001 and P=0.01, respectively. TET2 expression was also significantly reduced in AML compared to MDS (P=0.0007). MDS patients were stratified into low and high-risk disease, and we still observed a significant reduction in TET2 expression in high-risk (0.73, 0.17–7.25) when compared to low-risk (1.58; 0.48–21.30; P=0.02) patients, but no difference was noted between normal donors vs. low-risk MDS, and high-risk MDS vs. AML. In MDS cohort, the median overall survival was 14 months (range 1–83), increased TET2 expression was associated with a longer survival (HR, 0.44; 95% CI, 0.21–0.91, P=0.03), and, as expected, WHO high-risk disease was associated with a shorter survival (HR, 10.16; 95% CI, 3.06–33.72, P<0.001), even though the confidence interval (CI) was large. TET2 expression did not impact survival in our cohort of AML patients. The erythroid differentiation was effective in cells from normal donors and MDS patients, as demonstrated by the flow cytometry analyses of GPA and CD71. TET2 expression was significantly increased on day 12 of erythroid differentiation, P<0.05. On the other hand, DNMT3A expression was similar between normal donors (0.74; 0.22–1.53), MDS (0.78; 0.26–3.46) and AML (0.95, 0.15–6.46), and during erythroid differentiation, with no impact on survival.These data suggest that decreased TET2 expression may participate in leukemogenesis, and supports the participation of TET2 in the erythroid differentiation of MDS. DNMT3A was not differentially expressed in AML and MDS, indicating that the presence of mutations in this gene may be the predominant mechanism of changes in protein function. We thus suggest that decreased TET2 expression may explain the reduced levels of 5-hmc found in TET2 wild type patients, and may become a predictive marker for outcomes in MDS and other myeloid diseases. Further studies would be necessary to better elucidate the clinical relevance and biologic significance of our findings, and whether the decreased TET2 expression results in hypermethylation in these diseases.Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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.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.SIVA expression was significantly decreased in AML and ALL cells compared with normal hematopoietic cells (P<0.05), a reverse pattern of ANKHD1 expression, when compared with published data. Immunopreciptation and confocal analyses confirmed that ANKHD1 and SIVA interact and co-localize in the cytoplasm of leukemia cells. Functional studies revealed that SIVA and ANKHD1 have antagonistic effects on migration, Stathmin activation, and in vivo tumor growth. SIVA silencing resulted in a significantly increased cell migration, Stathmin activation (decreased Stathmin phosphorylation), and augmented in vivo tumor growth (P<0.05). On the other hand, ANKHD1 silencing resulted in a significantly decreased cell migration, Stathmin inactivation (increased Stathmin phosphorylation and alpha tubulin acetylation), and reduced in vivo tumor growth (P<0.05). Regarding apoptosis and proliferation, SIVA knockdown resulted in a significant decrease in apoptosis response to UV and daunorubicin induction and a downregulation of proapoptotic proteins p-JNK and BAX, an upregulation of the antiapoptotic protein BCL-XL, but no modulation was observed in proliferation and clonal growth in vitro. In contrast, ANKHD1 knockdown resulted in a significant decrease of proliferation and clonogenicity (P<0.05), but no changes were observed in apoptosis in vitro.Our data indicate SIVA as a tumor suppressor gene in leukemia cells, and SIVA downmodulation may contribute to the apoptosis resistance and chromosome instability. ANKHD1 may be an oncogene, and the upregulation of this protein in leukemia cells might lead to increased proliferation and generate chromosomal instability through increased Stathmin activation. The results suggest that ANKHD1 inhibits SIVA and restoration of SIVA expression or inhibition of ANKHD1 may be an attractive approach in leukemia.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

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Abstract 3836

Abstract 3804

No relevant conflicts of interest to declare.