IntroductionEarly Death (ED) remains challenging in newly diagnosed acute promyelocytic leukemia (APL), especially in developing countries. The clinical and laboratory profile at diagnosis were evaluated and causes and risk factors were investigated in adult APL patients.MethodA retrospective real-life analysis of 141 medical records was performed of patients diagnosed with APL between 2007 and 2018, whether they were treated with the IC-APL 2006 protocol or not. Risk factors were assessed by univariate and multivariate analysis.Main resultsOverall, 112 patients were included in the study. ED occurred in 22.3% of cases, surpassing clinical trial reports, with non-protocol-eligible patients presenting notably higher rates (60%), potentially due to their clinical status. Hemorrhage (60%) and infection (33.3%) were the leading causes of ED. Univariate analysis associated ED to the ECOG score; white blood cell (WBC) count; body mass index; levels of hemoglobin, albumin, uric acid, and creatinine, aPTT and INR and FLT3 mutations. Multivariate analysis identified ECOG score ≥2 and elevated WBC count as independent risk factors.ConclusionED remains a substantial challenge in APL, especially in real-world settings with hemorrhage and infection being the leading causes. ECOG status and WBC count emerged as independent risk factors, while age and platelet count lacked a 30-day prognostic correlation. Evaluating prognostic enhancement tools in controlled trials and real-life settings is pivotal to improving APL outcomes.

ABSTRACT:The introduction of all-trans retinoic acid combined with anthracyclines has significantly improved the outcomes for patients diagnosed with acute promyelocytic leukemia (APL), and this strategy remains the standard of care in countries in which arsenic trioxide is not affordable. However, data from national registries and real-world databases indicate that low- and middle-income countries (LMIC) still face disappointing results, mainly because of high induction mortality and suboptimal management of complications. The American Society of Hematology established the International Consortium on Acute Leukemias (ICAL) to address this challenge through international clinical networking. Here, we present the findings from the International Consortium on Acute Promyelocytic Leukemia study involving 806 patients with APL recruited from 2005 to 2020 in Brazil, Chile, Paraguay, Peru, and Uruguay. The induction mortality rate has notably decreased to 14.6% compared with the pre-ICAL rate of 32%. Multivariable logistic regression analysis revealed as factors associated with induction death: age of ≥40 years, Eastern Cooperative Oncology Group performance status score of 3, high-risk status based on the Programa Español de Tratamiento en Hematologia/Gruppo Italiano Malattie EMatologiche dell'Adulto classification, albumin level of ≤3.5 g/dL, bcr3 PML/RARA isoform, the interval between presenting symptoms to diagnosis exceeding 48 hours, and the occurrence of central nervous system and pulmonary bleeding. With a median follow-up of 53 months, the estimated 4-year overall survival rate is 81%, the 4-year disease-free survival rate is 80%, and the 4-year cumulative incidence of relapse rate is 15%. These results parallel those observed in studies conducted in high-income countries, highlighting the long-term effectiveness of developing clinical networks to improve clinical care and infrastructure in LMIC.

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17–11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

The SLIT-ROBO axis plays an important role in normal stem-cell biology, with possible repercussions on cancer stem cell emergence. Although the Promyelocytic Leukemia (PML) protein can regulate SLIT2 expression in the central nervous system, little is known about SLIT2 in acute promyelocytic leukemia. Hence, we aimed to investigate the levels of SLIT2 in acute promyelocytic leukemia (APL) and assess its biological activity in vitro and in vivo. Our analysis indicated that blasts with SLIT2high transcript levels were associated with cell cycle arrest, while SLIT2low APL blasts displayed a more stem-cell like phenotype. In a retrospective analysis using a cohort of patients treated with all-trans retinoic acid (ATRA) and anthracyclines, high SLIT2 expression was correlated with reduced leukocyte count (p = 0.024), and independently associated with improved overall survival (hazard ratio: 0.94; 95% confidence interval: 0.92–0.97; p < 0.001). Functionally, SLIT2-knockdown in primary APL blasts and cell lines led to increased cell proliferation and resistance to arsenic trioxide induced apoptosis. Finally, in vivo transplant of Slit2-silenced primary APL blasts promoted increased leukocyte count (p = 0.001) and decreased overall survival (p = 0.002) compared with the control. In summary, our data highlight the tumor suppressive function of SLIT2 in APL and its deteriorating effects on disease progression when downregulated.

Abstract: A faculty training workshop on problem-based learning (PBL) methods was provided to educators at a public medical school in Brazil. Wprkshop activities were designed to address specific knowledge, skill and attitudinal objectives that had been defined beforehand. Participants completed a structured questionnaire before and after the workshop to document attainment of the objectives. Twelve participants completed the 20-hour workshop, and questionnaire results. Though there were certain limitations on interpretation of the pre/post questionnaire, it does provide some assurance to program organizers and sponsors that objectives are attained. Participants' responses also help organizers improve their delivery techniques.

Introduction: Described as a well know marker of worse prognosis in acute myeloid leukemia (AML), MN1 overexpression has been associated with inv(16) or EVI1 overexpression (Heuser et al., Blood 2007). The promoter region of the MN1 gene has Retinoic Acid Response Elements (RAREs), and higher levels of MN1 expression have been associated with decreased response to retinoic acid (RA) in vitro. Nevertheless, in the context of acute promyelocytic leukemia, little is known about MN1 gene expression and functionality in vivo. Aims: Here, we investigated the effects of in vitro treatment with RA plus arsenic trioxide (ATO) in APL cell lines and primary blasts overexpressing MN1. Additionally, we quantified MN1 expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=2) and APL patients (age, 36-45y; n=2) were transduced with MN1 or empty vector (EV, control) to evaluate cell proliferation, differentiation and apoptosis. Confirmatory assays were performed using transduced NB4 and NB4R2 (RA-resistant) cell lines. After synchronization using double thymidine block, transduced cells were submitted to proliferation and clonogenic (treated with RA and ATO, as well) assays. To evaluate the apoptotic rate, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM each), for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) was evaluated based on the CD11b and CD11c surface levels. In addition, 116 patients (age, 18-82y; 51% males) with newly diagnosed APL enrolled in the ICAPL2006 study were included. To validate our data, Bootstrap resampling procedure with 1000 repetitions from the original database was performed to assess the model bias. Results: Primary APL cells transduced with MN1 (from TM/APL patients) presented higher proliferation rates compared to controls (P<.05). Similarly, the overexpression of MN1 in APL cell lines was associated with increased proliferation (P=.001) and clonogenicity (P<.05). Furthermore, NB4-MN1 cells are able to form colonies in the presence of RA (1 µM) (P<.01) but not under ATO (1 µM) treatment (P>.05), while NB4R2-MN1 cells were able to form colonies in the presence of ATO (P<.05). To investigate whether MN1 promotes resistance to drug-induced apoptosis, we treated lentivirally transduced cells with RA plus ATO for 72 hours. No differences were founded between the MN1-transduced and the EV cells (P>.05). In accordance with our results using primary APL samples, ATO treatment (alone or in combination with RA) does not modulate the drug-induced apoptosis in a time-dependent manner in NB4 and NB4R2-MN1 cells (P<.05). For NB4 cells, the differentiation rate was lower in MN1-expressing cells under RA alone or in combination with ATO for 48 hours (P<.05), although these effects were abrogated after 72 hours of RA and ATO treatment. In contrast, NB4R2-MN1 cells exhibited decreased differentiation rate at 48 and 72 hours in presence of RA alone or in combination with ATO, in comparison with EV cells (P<.05). In the clinical setting, the retrospective analysis of patients enrolled in the ICAPL2006 study revealed that the baseline characteristics were similar between patients with low and high MN1 levels. According to PETHEMA/GIMEMA criteria for predicting relapse, 34% and 46% of patients assigned to the low- and high-MN1 groups were deemed high-risk patients, respectively (P=.045). High MN1 expression was associated with lower 5-y Disease-Free Survival rates (74%; 95% CI: 69-91%)(HR: 2.61, 95% CI: 0.86-8.36) and Cumulative Incidence of Relapse (25%; 95% CI: 13-34%) (HR: 2.27, 95% CI: 0.75-6. 8). Bootstrap analysis for internal validation resulted in an AUC (0.63, 95% CI: 0.57-0.809) very similar to the original described data. Conclusion: We show that MN1 is relevant for RA-induced differentiation both in vitro and in vivo and may be involved in RA-resistance. Additionally, treatment with ATO circumvented the differentiation blockage in MN1 cells. Disclosures Pagnano: Sandoz: Consultancy; Pint Pharma: Consultancy; Abbvie: Consultancy. Tallman:International Conference in Leukemia: Honoraria; 14th Annual Miami Cancer Meeting: Honoraria; New Orleans Summer Cancer Conference: Honoraria; Indy Hematology Review: Honoraria; ADC Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding; Cellerant Therapeutics: Research Funding; Orsenix: Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Mayo Clinic: Honoraria; Rigel: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees, Research Funding; Salzberg Weill Cornall MSKCC Seminar in Hematologic Malignancies: Honoraria; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Oklahoma Medical Center: Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy; Danbury Hospital Tumor Board: Honoraria; Hematology Oncology of Indiana: Honoraria. Dillon:Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria. Heuser:Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Löwenberg:Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees.

Background: TP73 isoforms gained particular relevance in acute promyelocytic leukemia (APL) since Bernasola et al (JEM. 2004) demonstrated that TAp73 was directly regulated by the PML protein in the nuclear body. The isoforms differ in their transcriptional activity, with those lacking domains in the N-terminal part of the protein exerting a dominant negative effect on TP73 function. In a retrospective analysis of patients with APL treated in ICAPL study, Lucena-Araujo et al (Blood 2015) demonstrated the association between higher ΔNp73/TAp73 ratio values and poor clinical outcome. However,there is a diversity of TP73 isoforms and specially those lacking N-terminal domains (e.g.ΔEx2p73, ΔEx2-3p73 and ΔN'p73) may be relevant in APL genesis and therapy response. Aims: Here, we quantified transcript levels of TP73 N-terminal variants TAp73, ΔNp73, ΔEx2p73, ΔEx2-3p73 and ΔN'p73, as well as the C-terminal variants TP73αand TP73β, and determined whether there is a prognostic correlation. In addition, we evaluated the effect of ΔNp73overexpression on APL cell lines survival and differentiation in vitro and in vivo. Methods: Bone marrow (BM) samples from 98 patients (age, 18-74y) with newly diagnosed APL enrolled in the International Consortium on Acute Leukemia (ICAPL2006) were included. For comparison, BM mononuclear cells from 14 healthy donors (age, 18-60y) were also included. TP73 transcripts were determined by qPCR and using survival ROC curve analysis and the C-index we dichotomized patients into “low” and “high” expression. Proportional hazard model on overall survival (OS) and disease-free survival (DFS) was performed to evaluate prognosis. In addition, empty vector (EV) or ΔNp73α was transduced in NB4 and NB4R2 (RA-resistant) APL cell lines using a lentivirus system. The apoptosis rate was evaluated in both cell lines upon ATO (1 μM) treatment for 24, 48 and 72h. ΔNp73α, cleaved caspase 3 and Bcl-2 protein abundance was detected by western blotting. Granulocytic differentiation induced by RA-treatment (1 μM) alone or in combination with ATO (1 μM) for 72 h was assessed by CD11b surface levels. Finally, lethally irradiated 8-12 week old C57/BL6 Boy (CD45.1) were transplanted with hCG-PMLRARa Blasts (CD45.2) ΔNp73α or EV transduced (ΔNp73α group=16 and EV group=12). Results: Compared to normal BM, APL patients presented higher levels of ΔNp73 (p=.004), ΔEx2p73 (p=.008), and TP73β (p<.001) mRNA expression and lower of TAp73 (p<.001). ΔNp73 and ΔEx2p73 expression levels were directly correlated with TP73β (rho=.52 and .54, respectively), and TAp73 with TP73α (rho = .6). The 5y OS rate was significantly higher in patients with lower expression of ΔEx2p73 (78% vs. 97%, p<.05) and in patients with ΔEx2p73/TAp73 ratio values below the cut-off (77% vs. 100%, P<.04), and both variables were independent considering sex, age and leukocytes as confounders (HR=8, 95% CI:1.1-60.8 and HR=15.3, 95% CI: 2.1-1962,respectively). Furthermore, levels of ΔNp73 expression higher than the cut off were also associated with lower 5y OS rate (78% vs. 97%), establishing the fact that ΔNp73, ΔEx2p73 and TAp73 have similar patterns of expression and impact on APL treatment outcome. There was no association between TP73 isoform expression and DFS rate. Regarding the granulocytic differentiation, ΔNp73αoverexpression lead to RA-resistance in NB4 cells (p<.05), although the addition of ATO circumvented effect. As expected, ΔNp73αtransduced cells presented resistance to ATO induced apoptosis, which was detected at 48h (22.4 vs.49.8, p<.001) and 72h (47.3 vs.76.9, p=.032). We observed a partial clearance of ΔNp73α protein after 72h of ATO treatment, which may explain why ATO overcomes RA resistance induced by ΔNp73α, and why the apoptosis resistance to ATO was less evident after 72h compared to 48h. No significant difference in cleaved caspase 3 and Bcl-2 was detected during ATO treatment. No effect of ΔNp73αoverexpression was observed for NB4R2 cells. In vivo model showed no differences regarding peripheral blood counts, spleen weight (p>.05) or disease progression in ΔNp73α blasts compared to the control group (p=.095). Conclusions: Our results suggest that transcript levels of ΔNp73, ΔEx2p73 and TAp73 predict outcomes in APL patients treated with RA plus chemotherapy. Additionally, RA plus ATO combination therapy seemed to overcome the effect of ΔNp73 overexpression in vitro.

Digital Health (e-Health) is the use of the technologies of information and communication in the health area. We report the design and implementation of a course in e-Health for multi-professional postgraduate students. It was based on two key ideas: a blended-learning model and real-life spaces. The methodological triangulation approach included the educational planning of a course. In this work, we present and demonstrate the feasibility to use a blended-learning model to teach e-Health to postgraduate students based on interactive distributed learning spaces.

BACKGROUND:The establishment of regional development poles in the State of Pernambuco, Brazil was characterized by industrial expansion and consequent concerns about the increase in the occurrence of diseases, specifically those having long latency periods, as is the case of Chronic Myeloid Leukemia. METHODS:The study included 367 patients diagnosed with Chronic Myeloid Leukemia over a ten-year period at a reference treatment center. Records of patient charts and the TerraView software were used, respectively, for data collection and geographic mapping of the cases from the twelve established State development regions. RESULTS:A total incidence of 3.4 cases per 100,000 inhabitants was found, with a predominance of the disease among males, a median age of 47 years, a mestizo ethnicity, with elementary schooling and residence in urban area. Microregional incidence varied, but there was no significant variation in numbers over the years, and no relevant socio-environmental determinants were identified. CONCLUSION:The present study determined the incidence and characterized the spatial distribution of Chronic Myeloid Leukemia cases over a decade in a northeastern Brazilian state. The variation in the incidence rate by region of development is compatible with a homogeneous distribution of the cases. The work is a baseline study to be used for present and future analyses of the impact of the state economic development poles and the occurrence of this chronic malignant disease.

Background: The Slit-Robo pathway has been shown to participate in the pathogenesis of several malignant diseases in addition to its physiologic role during the development of the central nervous system (CNS). However, the relevance of the Slit-Robo pathway in acute promyelocytic leukemia (APL) is presently unknown. We investigated the status of the Slit-Robo pathway in APL following the recent demonstration by Amodeo et al (Cell Rep. 2017) that the PML protein induces neural stem/progenitor cells migration by inhibiting the SLIT2 gene, which was associated with an increased presence of H3K27me3 in the SLIT2 promotor region. Moreover, sensitivity towards the PML-targeting drug arsenic trioxide in primary glioblastoma cells was shown to be regulated by the PML/SLIT axis. Aims: Here, we quantified SLIT2 transcript levels in bone marrow (BM) samples from APL patients and healthy subjects and determined whether they are associated with clinical and laboratory features at diagnosis and treatment outcomes. In addition, we evaluated the effect of increased SLIT2 protein on leukemic cell survival, proliferation and clonogenecity. Methods: Cell cycle distribution, proliferation index (Ki-67/proliferation curve), colony formation and rate of apoptotic cells (annexin V/PI) were evaluated in both APL cell lines NB4 (ATRA-sensitive), NB4R2 (ATRA- resistant) and four primary APL samples following the treatment with the human recombinant SLIT2 protein (50 ng/ml) for 24 to 72 hours. In addition, 88 patients (age, 18-73y) with newly diagnosed APL enrolled in the International Consortium on Acute Leukemia (ICAL) study - ICAPL2006 were included. For comparison, BM mononuclear cells from five healthy volunteers (age, 18-60y) were also included. SLIT2 transcripts was determined by qPCR and expressed as comparative Ct method. Patients were dichotomized into “low” and “high” SLIT2 expression groups using a cut off value of 1.53 based on survival receiver operating characteristic (ROC) curve and the C index analyses. The following parameters were used to evaluate treatment outcome: complete hematological remission (CHR); 5-year Disease-Free Survival (DFS) and 5-year Overall Survival (OS) rates. Results: At the end of the sixth day, in vitro treatment with SLIT2 significantly reduced the proliferation rate (P=.01) and clonogenicity (P=.01) in primary APL samples, while significantly increasing the apoptotic rate after 72 hours treatment with SLIT2 (P=.03). In accordance, SLIT2 treatment decreased cell proliferation and clonogenicity (all P<.01) and increased the drug-induced apoptosis in a time-dependent manner in NB4 and NB4R2 cells (P<.01). Cell cycle analysis revealed that the cell cycle was arrested in the S-phase (P=.001) with a reduction of the G2/M phase (P=.001) in NB4 after 72 hours of treatment but no such impact was observed on NB4R2. In the clinical setting, patients with APL had a higher-than-normal expression of SLIT2 (6.7-fold higher than BMMC; P=.01). Baseline characteristics were similar between patients with low and high SLIT2 levels, except for a higher white blood cell count in patients with low SLIT2 expression (P=.024). Overall, 80/94 (85%) patients achieved complete hematological remission (CR). SLIT2 transcript levels had no impact on CR rate (P=.099). The estimated 5y DFS and the CIR rates were 87% (95% CI: 80-92%) and 12% (95% CI: 7-17%), respectively. Patients with low SLIT2 expression presented a lower 5y DFS rate (79%, 95% CI: 48-92%) than those with high SLIT2 expression (96%, 95% CI: 64-97%) (P=.026). In addition, patients with low SLIT2 expression exhibited an enhanced CIR rate compared to high SLIT2 expression (21% vs 11%) (P=.03). With a median follow up of 32 months (1-101 months), the estimated 5y OS rate was 79% (95% CI: 72-84%). Patients with lower SLIT2 expression had a lower 5y OS rate (68%, 95% CI: 45-83%) compared to those with higher SLIT2 expression (84%, 95% CI: 73-91%) (P=.008). This result was consistent with the multivariable proportional hazards analysis (HR: 1.02; 95% CI: 1.01-1.03; P=.001). Conclusion: Our results suggest that SLIT2 transcript levels may predict outcomes in APL patients treated with ATRA and anthracycline-based chemotherapy. Our data show that SLIT2 treatment represses APL cell growth, colony formation and induces apoptosis in vitro and we are currently assessing the role of the Slit-Robo pathway in the hCG-PML/RARA transgenic mouse model.

Introduction: FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P<.05), compared to only 0.5-fold reduction rate using ATRA alone. In the clinical setting, APL patients expressed a lower absolute level of FOXO3A than normal BMMC (P<.001). These results were corroborated in an external validation cohort (P=.004). The retrospective analysis of patients enrolled in the ICAPL2006 study revealed that the baseline characteristics were similar between patients with low and high FOXO3A levels. Low FOXO3A expression was associated with lower DFS (84%; 95% CI: 63-94%)(HR: 1.25, 95% CI: 0.64-10.4) and OS (76%; 95% CI: 63-84%) (HR: 1.43, 95% CI: 0.12-1.48). The overexpression of FOXO3A in APL cell lines was associated with reduced basal cell viability (P=.02), clonogenicity (P=.001), and proliferation (P=.001) in both APL cell lines. Subsequent cell cycle analysis showed no significant difference between FOXO3A-cells and controls in normal conditions and when treated with ATRA. Using a nonlinear regression analysis, IC50 for ATO was significantly lower for NB4-FOXO3A cells (0.27μM) than control (2.27µM), with no corresponding response for NB4R2 cell line (2µM for empty vector vs 1.46μM for NB4R2 FOXO3A). NB4 (IC50: 14mM) and NB4R2 (IC50: 4.2mM) FOXO3A cells presented higher sensibility to metformin treatment versus control group (IC50: 23mM for NB4; IC50: 22mM for NB4R2). For ATRA treatment alone, only NB4-FOXO3A presented increased sensitivity to ATRA (P=.001). In accordance, ATRA treatment (alone or in combination with metformin) increased the drug-induced apoptosis in a time-dependent manner (P<.05) in NB4-FOXO3A cells, but had no effect on NB4R2-FOXO3A, which presented increased apoptosis rate only with metformin treatment. The differentiation rate was higher in FOXO3A-expressing cells (P<.05). Conclusion: Based on clinical and functional data, the FOXO3A pathway could be an interesting target to overcome ATRA resistance in APL in offsetting where ATO is unavailable in low- and middle-income countries.

RESUMOINTRODUÇÃO: O câncer infantojuvenil vem se apresentando como a segunda causa de óbito na população entre 0 e 19 anos no Brasil, atrás apenas das causas externas. As tecnologias de informação e comunicação, especificamente, a telessaúde, revela-se uma mola propulsora na triagem de suspeitas de neoplasias malignas, além de promover a integração da equipe da atenção primária à saúde e a do centro de referência, diminuindo distâncias, e promovendo educação continuada igualitária.OBJETIVO: Associar os resultados do pré e pós-testes e descrever as teleconsultorias enviadas após a intervenção do projeto FIQUE ATENTO: PODE SER CÂNCER: A telessaúde como ferramenta para a suspeição precoce do câncer infantojuvenil, realizado com profissionais da atenção primária à saúde em Recife-Pernambuco.MÉTODO: Trata-se de um estudo quase-experimental sem grupo controle, descritivo, com abordagem quantitativa. Realizado nos anos de 2015 e 2016, a partir da análise de dados secundários da Plataforma de Telessaúde HealthNET da RedeNutes, do Núcleo de Telessaúde da Universidade Federal de Pernambuco. Utilizou-se o software EpiInfo 7.2 como suporte estatístico para o cálculo das frequências absolutas e relativas, assim como para as medidas de associação. Adotou-se o p-valor menor que 0,05 para o cálculo de significância estatística. O projeto foi aprovado sob o número do CAAE 50707515.7.0000.5208.RESULTADOS: Os profissionais, após a intervenção, mostraram maior desenvolvimento com relação aos conhecimentos obtidos sobre epidemiologia (p<0,001), e sinais e sintomas das neoplasias infantojuvenis (p<0,001). Foram geradas oito teleconsultorias com tempo médio de resposta de 53,32 horas, em que 50% (4) foram casos clínicos e tiveram seus encaminhamentos qualificados, e nenhum precisou ser regulado à unidade de referência.CONCLUSÃO: A telessaúde mostra-se como uma ferramenta com potencial de educação permanente, contribuindo para o diagnóstico precoce do câncer infantojuvenil, aumentando as chances de cura e sobrevida. Palavras-chave: Saúde da Criança; Saúde do Adolescente; Neoplasias; Telessaúde; Atenção primária à saúde.

By combining the analysis of mutations with aberrant expression of genes previously related to poorer prognosis in both acute promyelocytic leukemia (APL) and acute myeloid leukemia, we arrived at an integrative score in APL (ISAPL) and demonstrated its relationship with clinical outcomes of patients treated with all-trans retinoic acid (ATRA) in combination with anthracycline-based chemotherapy. Based on fms-like tyrosine kinase-3-internal tandem duplication mutational status; the Delta Np73/TAp73 expression ratio; and ID1, BAALC, ERG, and KMT2E gene expression levels, we modeled ISAPL in 159 patients (median ISAPL score, 3; range, 0-10). ISAPL modeling identified 2 distinct groups of patients, with significant differences in early mortality (P <.001), remission (P = .004), overall survival (P < .001), cumulative incidence of relapse (P = .028), disease-free survival (P = .03), and event-free survival (P < .001). These data were internally validated by using a bootstrap resampling procedure. At least for patients treated with ATRA and anthracycline-based chemotherapy, ISAPL modeling may identify those who need to be treated differently to maximize their chances for a cure.

Although overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is associated with primary resistant disease and shorter relapse-free, disease-free, and overall survival in different subsets of acute myeloid leukemia (AML), little is known about its clinical impact in acute promyelocytic leukemia (APL). Using real-time reverse transcriptase polymerase chain reaction, we showed that BAALC expression is significantly lower in APL compared with other subsets of AML (P < .001). We also demonstrated that BAALC overexpression was associated with shorter disease-free survival (DFS) (hazard ratio [HR], 4.43; 95% confidence interval [CI], 1.29-15.2; P = .018) in 221 consecutive patients (median age, 35 years; range, 18-82 years) with newly diagnosed APL homogeneously treated with all-trans retinoic acid and anthracycline-based chemotherapy. Cox proportional hazard modeling showed that BAALC overexpression was independently associated with shorter DFS in the total cohort (HR, 5.26; 95% CI, 1.52-18.2; P = .009) and in patients with high-risk disease (ie, those with initial leukocyte counts >10 × 109/L) (HR, 5.3; 95% CI, 1.14-24.5; P = .033). We conclude that BAALC expression could be useful for refining risk stratification in APL, although this needs to be confirmed in independent cohorts.

Introduction : Stathmin 1 is a microtubule destabilizer protein involved in the proliferation and differentiation of early hematopoietic progenitors. Aberrant Stathmin 1 expression has been reported in hematological malignancies. Stathmin 1 silencing reduces cell proliferation and clonogenicity of leukemia cell lines. A previous study identified that the PML-RARα fusion protein, which is necessary to acute promyelocytic leukemia (APL) genesis, positively regulates Stathmin 1 at transcription and protein activity levels. However, Stathmin 1 expression and its impact on prognosis and leukemia cell biology have rarely been studied in APL. Objectives : To evaluate Stathmin 1 expression and its association with laboratory and clinical parameters in a cohort of APL patients. To investigate Stathmin 1 expression and activity upon all-trans retinoic acid (ATRA) treatment and during cell cycle in the PML-RARα cell lines. Finally, to phenocopy the effects of Stathmin 1 inhibition using the pharmacological microtubule stabilizing agent paclitaxel. Methods : Bone marrow samples from 121 APL patients enrolled in the International Consortium on Acute Promyelocytic Leukemia (IC-APL) at diagnosis and 22 healthy donors were included. Patients were treated with ATRA and chemotherapy as described elsewhere. Patients were dichotomized according to Stathmin 1 expression using the median as cut-off. The PML-RARα cell lines, NB4 (ATRA-sensible) and NB4-R2 (ATRA-resistant) were used for functional assays. Cell cycle was synchronized by double thymidine block. For dose-response curves (MTT), PML-RARα cells were treated with graded concentrations of paclitaxel and IC50 values were calculated using a nonlinear regression analysis. For apoptosis assay (annexin V/PI), PML-RARα cells were treated with ATRA (1 µM) and/or IC50 paclitaxel dose for 72 hours. Gene and protein expressions were evaluated by qPCR and Western blot, and microtubule networks by confocal microscopy. For statistical analyses, Fisher's exact test or Chi-square test were used to compare categorical variables. Mann-Whitney test, t Student test or ANOVA test and Bonferroni post-test were used to compare continuous variables. Log-rank test and Cox regression were used for survival analyzes. P -value <0.05 was considerate statistically significant. Results : Stathmin 1 transcripts were significantly increased in bone marrow samples from APL patients compared with healthy donors (median 1.21 [range 0.02-4.62] versus (vs.) 0.66 [0.01-1.71]; p <0.01). Stathmin 1 levels did not impact on disease-free survival, overall survival and complete remission rates of APL patients. No relevant association between Stathmin 1 expression and clinical and laboratory parameters were observed. In NB4 cells, but not in NB4-R2, ATRA induced granulocytic differentiation, associated with a prominent reduction of Stathmin 1 gene and protein expression (p <0.05). Paradoxically, ATRA-induced granulocytic differentiation strongly reduced α-tubulin acetylation (microtubule stability marker) in NB4 cells. Using thymidine-synchronized cells, we observed that Stathmin 1 phosphorylation at serine 16 (an inhibitory site) was predominant in pool of mitosis-enriched cells, and its phosphorylation was marked reduced in pool of G1-enriched cells. NB4 and NB4-R2 cells presented high sensibility to paclitaxel treatment (IC50 of 3.6 nM and 1.8 nM, respectively). In NB4-R2 cells, but not in NB4 cells, paclitaxel increased the number of apoptotic cells after 72 hours of ATRA treatment; increased annexin V and caspase 3 cleavage (p <0.05). Paclitaxel treatment increased acetyl-α-tubulin, y-H2AX and CDKN1A levels. Conclusions : Stathmin 1 is highly expressed, but does not impact clinical outcomes in APL patients. Stathmin 1 reduces during ATRA-induced cell differentiation. Stathmin 1 activity is reduced during mitosis (as demonstrate by increased p-Stathmin 1S16) and increases post-mitosis (reduced p-Stathmin 1S16), indicating that this protein participates in microtubule dynamics and cell cycle progression in PML-RARα cells. Paclitaxel was an efficient pharmacological agent in inducing microtubule stabilization, reducing cell viability, increasing mitotic catastrophe as monotherapy or in combination with ATRA, in PML-RARα cells, including ATRA-resistant cells. Our study provides new insights on Stathmin 1 and microtubule dynamics in APL.

In chronic myeloid leukemia (CML) two main types of messenger RNA (e14a2 and e13a2) can be produced by BCR-ABL1 gene rearrangement. Due to conflicting results, the clinical value of these transcripts remains controversial. The aim of this study was to identify associations of e14a2 and e13a2 transcripts with laboratory variables and also the response to treatment. This study included 203 adult patients with CML treated with Imatinib as first-line drug in a reference hematology center in Northeast Brazil. Clinical and laboratory data were obtained after informed consent. Samples were collected for RNA extraction and analyzed by reverse transcription-polymerase chain reaction (PCR), according to the international protocol BIOMED-1. The LeukemiaNet 2013 criteria were used to establish the molecular response. The frequency distribution of the BCR-ABL1 transcripts was e14a2 (64%), e13a2 (34%), and double positives (2%). The results showed a statistically significant association of the e14a2 transcript type with thrombocytosis (P = 0.0005) and the e13a2 with higher leukocyte count (P = 0.0491). In a subgroup of 44 patients, the molecular response to treatment with Imatinib was assessed by quantitative PCR at 3 months (BCR-ABL1 ≤ 10%), 6 months (BCR-ABL1 ≤ 1%), or 12 months (BCR-ABL1 ≤ 0.1%). Although patients with the transcript e14a2 showed higher frequency of good responses than patients with the transcript e13a2, this difference was not statistically significant. In agreement with published data, our results showed association of the BCR-ABL1 transcript e14a2 with thrombocytosis and the BCR-ABL1 transcript e13a2 with higher leukocytosis in patients with chronic myeloid leukemia.