Lorena Lobo de Figueiredo-Pontes

Determination of P‐glycoprotein, MDR‐related protein 1, breast cancer resistance protein, and lung‐resistance protein expression in leukemic stem cells of acute myeloid leukemia

The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self‐renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets.

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

The presence of CD56/CD16 in T-cell acute lymphoblastic leukaemia correlates with the expression of cytotoxic molecules and is associated with worse response to treatment.

Some cases of T‐cell acute lymphoblastic leukaemia (ALL) express markers found in natural‐killer (NK) cells, such as CD56 and CD16. Out of 84 T‐cell ALL cases diagnosed at our Institution, CD56 and/or CD16 was detected in 24 (28·5%), which we designated T/NK‐ALL group. Clinical features, laboratory characteristics, survival and expression of cytotoxic molecules were compared in T/NK‐ALL and T‐ALL patients. Significant differences were observed regarding age (24·9 vs. 16·4 years in T/NK‐ALL and T‐ALL, respectively, P = 0·006) and platelet counts (177 × 109/l vs. 75 × 109/l in T/NK‐ALL and T‐ALL, respectively, P = 0·03). Immunophenotypic analysis demonstrated that CD34, CD45RA and CD33 were more expressed in T/NK‐ALL patients, whereas CD8 and terminal deoxynucleotidyl transferase were more expressed in T‐ALL patients (P < 0·05). The mean overall survival (863 vs. 1869 d, P = 0·02) and disease‐free survival (855 vs. 2095 d, P = 0·002) were shorter in patients expressing CD56/CD16. However, multivariate analysis identified CD56/CD16 as an independent prognostic factor only for DFS. Cytotoxic molecules were highly expressed in T/NK‐ALL compared to T‐ALL. Perforin, granzyme B and TIA‐1 were detected in 12/17, 4/17 and 7/24 T/NK‐ALL patients and in 1/20, 0/20 and 1/20 T‐ALL respectively (P < 0·001, P = 0·036 and P = 0·054). Therefore, the presence of CD56/CD16 was associated with distinct clinical features and expression of cytotoxic molecules in the blasts.

The co-expression of PML/RAR alpha and AML1/ETO fusion genes is associated with ATRA resistance

patients are routinely monitored by measuring their serum immunoglobulin and urine light chains – a logical step would be to supplement, or replace, the urine assays with serum FLC measurements, as has already happened in many centres. Thus, the conclusion of Tate et al (2005) that larger prospective studies are needed before FLC measurement is ‘routinely applied to the monitoring of patients with IIMM’, is a generalization, drawn outside the context of the data they have presented. Serial FLC measurement is being undertaken in all patients in the current Medical Research Council Myeloma IX Study, which should provide further information on patients treated in a systematic way. Future studies may investigate clinical benefit further, perhaps with modification or change in treatment determined by FLC measurement.

Results of FLT3 mutation screening and correlations with immunophenotyping in 169 Brazilian patients with acute myeloid leukemia

A. R. Lucena-Araujo :D. L. Souza : F. M. de Oliveira : M. T. L. Benicio : L. L. Figueiredo-Pontes : B. A. Santana-Lemos :G. A. dos Santos : R. H. Jacomo : E. M. Rego (*) Hematology Division, Department of Internal Medicine, National Institute of Science and Technology on Cell Based Therapy, Medical School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil e-mail: emrego@hcrp.fmrp.usp.br

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 μmol/mL and < 6.0 μmol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL.

The effect of intravitreal ranibizumab on intraoperative bleeding during pars plana vitrectomy for diabetic traction retinal detachment.

Traction retinal detachment (TRD) is an important cause of visual impairment in patients with advanced proliferative diabetic retinopathy (PDR). It is treated surgically and involves removal of fibrovascular membranes and release of the vitreoretinal traction during pars plana vitrectomy (PPV), with special care taken to ensure haemostasis and avoid iatrogenic retinal breaks.1 Based on the promising results using bevacizumab before PPV,2–4 we evaluated the effect of another humanised anti-VEGF-A drug, intravitreal ranibizumab (IVR), on reducing intraoperative bleeding during 23-gauge PPV in 19 consecutive patients with macula-involving TRD of up to 3 months duration secondary to PDR and best-corrected visual acuity between 20/60 and 20/800. Patients were not enrolled if they had vitreous haemorrhage preventing detailed visualisation of the entire posterior retinal pole, previous intraocular surgery except for cataract surgery, any clinical condition that would impair the documentation of the ocular fundus, previous thromboembolic events, known clotting disorders or use of anticoagulant medications except aspirin, or significant and uncontrolled diseases that might interfere with study participation. Patients were randomly assigned to administration of either …

Halofuginone inhibits phosphorylation of SMAD-2 reducing angiogenesis and leukemia burden in an acute promyelocytic leukemia mouse model

BackgroundHalofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-β (TGF-β) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model.MethodsNOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 μg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells.ResultsHF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-β-signaling.ConclusionTaken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.

Philadelphia-negative myeloproliferative neoplasms as disorders marked by cytokine modulation

Background Cytokines are key immune mediators in physiological and disease processes, whose increased levels have been associated with the physiopathology of hematopoietic malignancies, such as myeloproliferative neoplasms. Methods This study examined the plasma cytokine profiles of patients with essential thrombocythemia, primary myelofibrosis, polycythemia vera and of healthy subjects, and analyzed correlations with JAK2 V617F status and clinical-hematological parameters. Results The proinflammatory cytokine levels were increased in myeloproliferative neoplasm patients, and the presence of the JAK2 V617F mutation was associated with high IP-10 levels in primary myelofibrosis patients. Conclusions Essential thrombocythemia, primary myelofibrosis, and polycythemia vera patients exhibited different patterns of cytokine production, as revealed by cytokine network correlations. Together, these findings suggest that augmented cytokine levels are associated with the physiopathology of myeloproliferative neoplasms.

Crosstalk between BCR-ABL and protease-activated receptor 1 (PAR1) suggests a novel target in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome, which generates the oncogene BCR-ABL1. Protease-activated receptor 1 (PAR1) is involved in tumor progression and angiogenesis. We have previously reported that PAR1 expression is elevated in human leukemias that display a more aggressive clinical behavior, including the blast crisis of CML. In this study, we analyzed the crosstalk between the oncoprotein BCR-ABL and PAR1 in CML. Leukemic cell lines transfected with the BCR-ABL1 oncogene showed significantly higher expression levels of PAR1 compared with that of wild-type counterparts. This phenomenon was reversed by treatment with tyrosine kinase inhibitors (TKIs). Conversely, treatment with the PAR1 antagonist SCH79797 inhibited BCR-ABL expression. The PAR1 antagonist induced apoptosis in a dose- and time-dependent manner. Higher vascular endothelial growth factor (VEGF) levels were observed in cells transfected with BCR-ABL1 than in their wild-type counterparts. VEGF expression was strongly inhibited after treatment with either TKIs or the PAR1 antagonist. Finally, we evaluated PAR1 expression in CML patients who were either in the blast or chronic phases and had either received TKI treatment or no treatment. A significant decrease in PAR1 expression was observed in treatment-responsive patients, as opposed to a significant increase in PAR1 expression levels in treatment-resistant patients. Patients classified as high risk according to the Sokal index showed higher PAR1 expression levels. Our results demonstrate the crosstalk between BCR-ABL and PAR1. These data may offer important insight into the development of new therapeutic strategies for CML.