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Introdução: A quantificação de linfócitos T, B e Natural Killer (NK) por citometria de fluxo é o confiável para avaliar a imunocompetência do organismo, crucial para a homeostase e defesa contra patógenos e células danificadas. Alterações nas populações de linfócitos no sangue periférico estão associadas a diversas doenças, incluindo imunodeficiências primárias e secundárias, doenças autoimunes, infecções e câncer. A citometria de fluxo, utilizando anticorpos monoclonais conjugados a fluorocromos, permite a detecção e quantificação dessas subpopulações celulares. Esta tecnologia está em constante avanço, com melhorias contínuas nos equipamentos e procedimentos laboratoriais que aumentam a eficiência e precisão das análises. No entanto, é crucial que os resultados das análises laboratoriais sejam comparáveis independentemente da metodologia e da instrumentação diagnóstica utilizadas. Objetivo/Justificativa: Este estudo teve como objetivo validar e comparar a consistência e a precisão dos resultados da análise de linfócitos T, B e NK utilizando os citômetros de fluxo BD FACSCanto II e BD FACSLyric. Essa validação é fundamental para assegurar que a introdução do novo citômetro BD FACSLyric não comprometa a precisão diagnóstica e o acompanhamento do tratamento clínico, mantendo a qualidade dos diagnósticos realizados. Métodos: Foi realizada uma pesquisa experimental utilizando amostras de sangue periférico de 20 pacientes. As amostras foram marcadas com anticorpos para detecção de linfócitos T, B e NK e suas subpopulações, adquiridas simultaneamente em ambos os citômetros de fluxo. A análise da porcentagem parcial das populações celulares foi realizada utilizando o software Infinicyt. Foram conduzidas análises de correlação e de Bland-Altman. Resultados/Discussão: Todos os coeficientes de correlação de Pearson (r) na Análise de Correlação mostraram-se muito próximos de 1, indicando uma correlação positiva perfeita entre as medições dos dois citômetros de fluxo, com valor de p < 0,001 em todos os casos, o que indica significância estatística. A análise de Bland-Altman revelou pequenas diferenças nas medições entre os equipamentos, demonstrando alta concordância entre as medições dos dois citômetros para a quantificação das células. Os resultados obtidos neste estudo são consistentes com a maioria dos estudos comparativos realizados em outros contextos, evidenciando forte correlação e excelente comparabilidade entre os resultados obtidos na avaliação das subpopulações de linfócitos pelos dois citômetros de fluxo analisados. Conclusão: Este estudo demonstrou que os equipamentos BD FACSCanto II e BD FACSLyric são capazes de fornecer resultados consistentes e comparáveis para a maioria das subpopulações analisadas. Esses achados são cruciais para a validação e padronização dos métodos de análise, especialmente em ambientes clínicos e de pesquisa.

A Leucemia Linfoide Aguda (LLA) é uma neoplasia hematológica caracterizada pela proliferação descontrolada de células progenitoras linfoides B ou T, comprometendo o processo de hematopoese. Representando mais de 80% das leucemias agudas, a LLA de linhagem B é responsável por aproximadamente 85% dos casos, sendo mais comum em crianças. A imunofenotipagem por citometria de fluxo multiparamétrica (CFMP) desempenha um papel crucial na identificação e caracterização das leucemias, permitindo a detecção precisa de marcadores de diferenciação celular. Compreender os perfis de imunofenotipagem em LLA-B pode orientar o desenvolvimento de novas terapias direcionadas. Portanto, o presente estudo teve como objetivo avaliar o perfil de pacientes com LLA-B utilizando a CFMP. A análise de coorte retrospectiva foi realizada por meio da revisão dos prontuários eletrônicos dos pacientes, onde foram coletados dados demográficos e resultados da CFMP no momento do diagnóstico. Este estudo retrospectivo analisou os perfis de imunofenotipagem em 116 pacientes com LLA-B, coletando dados demográficos e de CFMP ao diagnóstico ao longo de cinco anos (jan/2018 a jan/2023). A análise da expressão de CD45 revelou intensidades dim, dim/negativo e negativo em 57, 52 e 6 pacientes, respectivamente. Entre os subtipos de LLA-B, a positividade para CD34 foi associada ao subtipo Comum. Todos os pacientes apresentaram positividade para CD19, e outros marcadores comumente expressos, como CD79a (97,4%), CD22 (81,8%) e CD10 (83,6%), foram positivos na maioria dos casos, ressaltando a importância da identificação alternativa das células B leucêmicas para terapias alvo-específicas. A análise dos subtipos revelou uma prevalência do tipo Comum (75,7%), seguido pelo subtipo Pró-B (14,8%) e Pré-B (9,6%). Observou-se uma predominância do sexo masculino nos subtipos Pró-B e Comum. No subtipo Pré-B, 54,5% dos pacientes eram do sexo feminino, enquanto 45,5% eram do sexo masculino. No subtipo Pró-B, 64,7% dos pacientes eram adultos e 35,3% eram pediátricos. Para o subtipo Comum, 37,9% eram adultos e 62,1% eram crianças. No subtipo Pré-B, associado a uma maior maturação das células leucêmicas, houve uma incidência de 45,5% de adultos e 54,5% de crianças. A variabilidade na expressão de CD45 e a associação de CD34 com o subtipo Comum sublinham a necessidade de uma identificação precisa para o desenvolvimento de estratégias terapêuticas personalizadas. A capacidade da CFMP em detectar e caracterizar de maneira detalhada os marcadores de diferenciação celular oferece uma base sólida para a criação de terapias direcionadas. A interpretação dos resultados reforça a importância clínica da investigação dos marcadores por CFMP, fornecendo informações valiosas para o avanço das estratégias terapêuticas e sugerindo novas direções para pesquisas futuras. A continuidade da pesquisa na área é essencial para aprimorar o tratamento e melhorar os desfechos clínicos para os pacientes com LLA-B.

Acute Myeloid Leukemia (AML) is a severe hematopoietic disorder marked by uncontrolled proliferation of immature myeloid progenitors due to differentiation arrest, with significant immunophenotypic diversity. FLT3-ITD mutations’ prognostic impact varies with NPM1 mutation presence and the FLT3 allelic ratio. While cytogenetic and molecular features are well-established for risk stratification by European LeukemiaNet (2017 ELN), the role of leukemia arrest stages in prognosis remains unclear. This study classified the maturation arrest stages of leukemic blasts in a Brazilian AML cohort, analyzing its association with genetic risk and clinical data. Bone marrow from 148 de novo AML patients (Sep-2015-Jul-2024) was evaluated for FLT3 and NPM1 mutations, FLT3 allelic ratio, and immunophenotyping. MPO cytochemistry stains and phenotypic markers by flow cytometry defined six stages of leukemia arrest (SLA): Hematopoietic Stem Cells-like (HSC-L:CD34+ CD117±CD13-CD33-HLA-DR+MPO-); Multipotent Progenitors-like (MPP-L:CD34+CD117+CD13±CD33±HLA-DR+MPO-); Common Myeloid Progenitors-like (CMP-L:CD34±CD117+CD13+CD33± HLA-DR+MPO+); Granulocyte-Monocyte Progenitors-like (GMP-L:CD34±CD117±CD13±CD33+HLA-DR+MPO+); Monocyte Progenitors-like (MP-L:CD34-CD117±CD13+CD33+HLA-DR+MPO+); and Granulocyte Progenitors-like (GP-L:CD34-CD117±CD13+ CD33+HLA-DR-MPO+). SLA showed highest frequency in MP-L (n = 57, 38.5%) and GP-L (n = 53, 35.8%), followed by GMP-L (n = 23, 15.5%), CMP-L (n = 7, 4.7%), MPP-L (n = 6, 4.1%), and HSC-L (n = 2, 1.4%). Predominance of FLT3wt/NPM1mut patients (n = 80, 54.1%) was observed, with similar distribution among FLT3mut patients (FLT3mut /NPM1mut:n = 32, 21.6%; FLT3mut/NPM1wt:n = 36, 24.3%). CMP-L and GMP-L frequencies were increased in FLT3low/NPM1wt and FLT3high/NPM1wt vs. FLT3wt/NPM1mut (CMP-L:13.33%, 14.28% vs 1.25%, p < 0.05; GMP-L:40%, 42.85% vs 7.5%, p < 0.05). GP-L frequency was decreased in FLT3low/NPM1wt and FLT3 high/NPM1wt vs. FLT3wt/NPM1mut (0%, 9.52% vs 43.75%, p < 0.05). Response to treatment (CR/CRi, CR/CRiMRD+ and PD–Persistence disease) was evaluated after 1st and 2nd cycles of Induction of Remission (1IR, 2IR). GMP-L was increased in PD after 1IR (OR:6.09, 95% CI:1.22–30.19, p < 0.05). Immature (HSC+MPP+CMP) SLA was higher in MRD+ patients compared to MRD- (OR:6.81, 95% CI:0.86–47.05, p < 0.05). Overall survival (p = 0.81) and relapse-free survival (p = 0.27) analyses showed that GMP-L patients relapsed sooner. In 2017 ELN risk assessment, FLT3-ITD stratification was based on allelic ratio and NPM1 mutation status. A high FLT3-ITD allelic ratio with NPM1 mutation indicated intermediate risk, and with wild-type NPM1, it indicated unfavorable risk. The 2022 ELN guidelines classify FLT3-ITD alone as intermediate risk, regardless of NPM1 status. Mutated NPM1 is favorable only when FLT3-ITD is absent. Supporting these findings, FLT3 mutation, regardless of allelic ratio, in the absence of NPM1, led to immature SLA and increased GMP-L frequency. This SLA was linked to PD after the 1IR. Immature SLA significantly impacts prognosis with lower survival rates, higher relapse risks, and increased post-induction remission failure. Our findings enhance the understanding of the cell of origin in AML, and further investigation of the SLA-dependent mechanisms that contribute to leukemia resistance are warranted.

Objetivos: A autofagia foi recentemente postulada como um potencial mecanismo que contribui para o desenvolvimento de resistência aos inibidores de FLT3 (FLT3i) em leucemia mieloide aguda (LMA). A cloroquina é um medicamento seguro e de baixo custo e um dos seus efeitos é a inibição da autofagia. Aqui, investigamos o potencial efeito anti-neoplásico da cloroquina (CQ) associada aos FLT3i em modelos de LMA FLT3-ITD. Métodos/Resultados: Em modelos de LMA FLT3-ITD (células MOLM13 e MV411), o tratamento com FLT3i (PKC412 e AC220) aumentou significativamente o fluxo autofágico induzindo a formação de organelas vesiculares ácidas, conforme quantificado por citometria de fluxo e confirmado por microscopia de fluorescência, por meio do marcador de autofagia laranja de acridina. Observamos uma redução nos reguladores negativos da autofagia, evidenciada pela inibição da fosforilação em AKT (pSer473), mTOR (pSer2448) e seu alvo a jusante p70S6K (pThr421/Ser424), bem como uma redução em p-ULK1 (pSer757). O tratamento com FLT3i resultou na conversão de LC3B-I em LC3B-II, seguido pela degradação de LC3B-II e p62, indicando indução de autofagia. A combinação de FLT3i com CQ (10 μM) reduziu significativamente a viabilidade e sobrevivência celular. Para investigar se a CQ induziu a morte celular por meio de potenciais efeitos off target e não devido ao bloqueio da autofagia, realizamos um silenciamento induzido por shRNA (KD) do mediador de autofagia ATG5 em células FLT3-ITD. O tratamento com FLT3i em células MOLM13 ATG5-KD reduziu significativamente o IC50 para o PKC412 (ATG5-KD:17nM vs controle:31nM) e aumentou a indução de apoptose. Para investigar se a inibição da autofagia seria uma opção para células resistentes a FLT3i, geramos células MV411 resistentes a AC220 (MV411 QR). Células MV411 QR exibiram um nível basal mais alto de autofagia em comparação com células MV411 convencionais, indicado pelo aumento da formação de organelas vesiculares ácidas e níveis reduzidos de proteína de LC3B e p62. O tratamento com CQ reduziu efetivamente o IC50 para o AC220 (monoterapia:7,68nM vs combinação:5,3nM) e aumentou a morte celular, sugerindo seu uso para mediar a resistência ao FLT3i na LMA. Molecularmente, a análise proteica revelou aumento no efeito do AC220 quando associado com CQ em células MV411 QR, evidenciado pelo aumento de Caspase3 e pela redução de pSTAT5 e pP70S6K. Amostras de células primárias de LMA tratadas ex vivo mostraram que a combinação de FLT3i com CQ reduziu a proliferação e sobrevivência em células FLT3-ITD. Por fim, realizamos um estudo in vivo em modelo murino de xenoenxerto (células MOLM13, n = 5 por grupo). Os animais foram tratados com veículo, PKC412 (6 mg/Kg/dia), CQ (20 mg/Kg/dia) e combinação até o óbito. O tratamento combinado aumentou a sobrevida global e preservou a hematopoese saudável. Discussão/Conclusão: O tratamento com FLT3i desencadeia fluxo de autofagia, restringindo a atividade inibitória da droga. A eficácia antileucêmica do FLT3i pode ser potencializada pela inibição da autofagia com CQ, superando a resistência farmacológica. Apesar da CQ apresentar tolerância clínica favorável, é essencial considerar a heterogeneidade do paciente. Avaliar o perfil autofágico na LMA pode contribuir significativamente para estratégias de tratamento personalizadas.

Myelofibrosis (MF), driven by JAK/STAT signaling mutations in hematopoietic stem cells, has the worst survival rates among myeloproliferative neoplasms (MPNs), worsening from pre-fibrotic MF (PFMF) to overt MF (OMF) that often precedes acute myeloid leukemia. Natural Killer (NK) cells have phenotypical alterations that predominate in MF when compared to other MPNs, suggesting that the immune content may contribute to disease burden. We hypothesized that the cytotoxic immune cell profile, along with fibrosis and mutations, drives MF progression. Aiming test this, peripheral blood mononuclear cells (PBMC) from 9 PFMF and 9 OMF, and 9 healthy donors (controls: CT) were analyzed by flow cytometry to profile: NK cell frequency and subtypes (CD45hiCD3-CD19-CD56brightCD16-; CD45hiCD3-CD19-CD56dimCD16+; NK maturation [CD11b-CD27- tolerant (DN), CD27+CD11b- immature secretory (IS), CD27+CD11b+ mature secretory (MS), CD11b+CD27- cytotoxic]; T lymphocytes (CD45hiCD3+) frequency and subtypes (CD4, CD8) and maturation [CD45RO-CD45RA+CD27+ Naïve (N), CD45RO+CD45RA+CD27+ intermediate (INT), CD45RO+CD45RA+CD27- Central Effector (CE), CD45RO+CD45RA-CD27+ Central Memory (CM)]. Immune evasion was assessed by CD62L, and Treg cells (CD4+CD25hiCD127lowFOxp3+) were also quantified. NK cytotoxic capacity was measured by quantifying dead/K562 target cells and NK degranulation (CD107a) after PBMC co-culture. Top of FormBottom of Form Total NK frequency was similar in MF vs CT or OMF vs PFMF. CD56bright (CT 3.4 ± 2.6%, MF 17.7 ± 17.9%, p < 0.05) were increased and CD56dim (CT 82.7 ± 7.6%, MF 53.4 ± 25%, p < 0.05) decreased in MF vs CT. MF showed higher DN (CT 4.5 ± 3.5%, MF 13.9 ± 8.7%, p < 0.05) and IS (CT 0.2 ± 0.3%, MF 1.2 ± 1.2%, p < 0.05), and lower cytotoxic NK cells (CT 92.1 ± 3.8%, MF 77.3 ± 11.2%, p < 0.05). When compared to PFMF, OMF presented higher frequency of cytotoxic NK cells (PFMF 72.2 ± 12%, OMF 82.4 ± 7.9%, p = 0.09). In agreement, reduced NK cytotoxic capacity was observed despite no degranulation changing in MF (death: CT 81.8 ± 4.2%, MF 30.1 ± 7.76%, p < 0.05) and slightly increased cytotoxicity (PFMF 28 ± 6.4%, OMF 31.4 ± 9.9%, p = 0.7) and degranulation (PFMF 10.1 ± 8.8%, OMF 32.17 ± 9.9%, p = 0.1) accompanied fibrosis. No changes in total T lymphocytes or CD4/CD8 frequencies were seen between CT and MF or PFMF and OMF. However, INT and CE reduced in MF (CT 19.8 ± 9.9%, MF 7.3 ± 12.2%, p < 0.05; CT 19.1 ± 16.4%, MF 8.1 ± 13.8%, p < 0.05) and CM decreased in OMF (PFMF 18.9 ± 11.9%, OMF 10.6 ± 9.5%, p < 0.05). CD62L+ CM decreased in MF (CT 21.5 ± 12.8%, MF 6.8 ± 6.8%, p < 0.05) and in OMF (PFMF 9.4 ± 8%, OMF 3.9 ± 3.7%, p = 0.2). Treg frequency decreased in MF (CT 1.9 ± 1.7%, MF 0.25 ± 0.22%, p < 0.05) but increased in OMF (PFMF 0.2 ± 0.13%, OMF 0.3 ± 0.2%, p = 0.5). More advanced MF has a higher degree of fibrosis, which leads to an inflammatory profile. This, in turn, may activate NK cells and result in a more mature but cytotoxic-deficient profile probably due to exhaustion. On the other hand, increased Treg cells found in OMF negatively regulate cytotoxic T cells and its memory compartment, specifically leading to immune evasion via a decrease in CD62L+ CM (associated with better anti-tumor response). The results shed light on the mechanisms involved in MF progression, suggesting that changes in the maturation profile of cytotoxic cells is associated with disease progression, thus inspiring new therapies that target antitumoral cells from the leukemic environment.

Patients with Chronic Myeloid Leukemia (CML) who achieve sustained deep molecular response (DMR) to Tyrosine Kinase Inhibitors (TKIs) are eligible for treatment discontinuation, but only 50% of those achieve long-term treatment-free remission (TFR). Current biomarkers that predict resistance to TKIs as well as loss of the molecular response after TKI interruption have not been defined. We hypothesized that antitumoral immunity mediated by Natural Killer (NK) and T cells may contribute to TFR success and result in distinct responses during treatment and after discontinuation. NK and T cells phenotype were correlated with BCR::ABL1 kinetics and TFR success in a cohort of CML Brazilian patients included in the DES-CML study (Study of treatment discontinuation in CML treated at the Unified Health System), multicenter, prospective, open-label, single-arm, phase 2, non-randomized, ongoing trial, with current data from two Brazilian centers. Using flow cytometry, we evaluated frequency, subtypes, maturation, and receptors expression of NK and T cells from diagnosis to TKI discontinuation. Peripheral blood samples from 20 healthy controls and 68 CML patients were analyzed at different time points: 14 diagnosis (DX), 13 Imatinib failure (F), 7 major molecular response (MMR), 9 DMR, 20 TFR and 5 TFR loss (TFRL). Patients in discontinuation cohort had TKI dose reduced to 50% for 6 months before total suspension. NK cell subtypes were defined as secretory (CD56brightCD16-) or cytotoxic (CD56dimCD16+). We assessed NK maturation markers (CD57 and NKp80), activation (NKG2D,NKp46,DNAM-1), and inhibition receptors (KIR2DL1,TIGIT,NKG2A). PD-1 checkpoint inhibitor was assessed in CD8 T cells. The frequencies of cytotoxic (p < 0.05) and mature (CD57+ and NKp80 p < 0.05) NK cells as well as NKG2D and DNAM-1 (p < 0.05) were lower in the DX, F, and TFRL groups as compared to patients under TKI treatment with DMR, TFR, and healthy individuals. Patients in TFR exhibited a distinct immune profile from TFRL patients that were not influenced by gender, TKI drug, ELTS or SOKAL score. TFR patients presented higher frequency of cytotoxic (p < 0.05) and mature (CD57+ p < 0.05) NK cells, and also NKG2D, DNAM-1,NKp46 (p < 0.05) compared with TFRL. Frequency of PD-1 (p < 0.05) in T cells was decreased in TFR. At the TKI dose reduction timepoint,TFRL patients already had decreased frequency of cytotoxic (p < 0.05) and mature (CD57+ p < 0.05) NK cells as well as of the activating receptor NKG2D (p < 0.05) compared with TFR cases. Conversely, T-cell PD-1 (p < 0.05) expression was higher in TFRL than in TFR patients at the dose reduction point. In conclusion, our study indicates that impaired NK cell function and PD-1 T-cell inhibition are major contributors to molecular responses in CML. Patients with sustained TFR exhibit higher frequencies of cytotoxic, mature and activated NK cells, along with lower PD-1 expression in T cells. These profile contrasts with patients who have persistent disease or lose molecular response post-treatment suspension, who demonstrate impaired NK cell function and increased PD-1 expression. Monitoring parameters such as mature NK content and activating receptors, particularly NKG2D, and PD-1 expression during the de-escalation phase of TKI potentially predicts TFR relapse and can be useful for decision-making in TKI interruption.

Introdução: Demonstramos previamente um comprometimento funcional das células NK no modelo murino JAK2V617F e células do sangue periférico de pacientes com Neoplasias Mieloproliferativas (NMPs), destacando-se o aumento da frequência de células NK imaturas e redução da citotoxicidade da fração mais madura CD27−CD11b+. Não se sabe se as células iniciadoras das NMPs são intrinsecamente resistentes à imunidade tumoral ou se elas são capazes de modificar as células NK do microambiente leucêmico, tornando-as disfuncionais. Objetivo: Para esclarecer estes mecanismos, o presente trabalho se propôs a modelar a exposição leucêmica de células imunes normais e verificar suas modificações utilizando um modelo de transplante quimérfico de NMP Jak2V617F (Jak2VF). Métodos: Utilizamos um modelo de transplante murino com condicionamento não mieloablativo (irradiação subletal única de 400cGy) de modo a conservar em média 50% das células do receptor. Foram injetadas 5 x 106 células de medula óssea total de animais Jak2VF (CD45.2) ou controles Jak2WT (CD45.2), por via retrorbital, em animais selvagens de mesma linhagem (C57BL/6, CD45.1, N = 6 por grupo). A enxertia foi avaliada por citometria de fluxo de acordo com a proporção de células CD45.2/CD45.1+CD45.2. Após a confirmação da enxertia, as células NK expostas à leucemia ou à medula óssea normal foram avaliadas quanto à frequência, subtipos funcionais e receptores. Resultados: A frequência de células NK expostas às células Jak2VF se mostrou menor do que as células NK expostas às células Jak2WT (Jak2WT 44,05%, -24,29 ±2,856 vs Jak2VF 19,76%, -30,75 ±-17,83, p < 0,0001). Quanto aos subtipos funcionais, as frações imaturas secretórias (Jak2WT 44,32%, -13,62 ± 5,703 vs Jak2VF 30,70, -26,52 ±-0,7157, p = 0,0407) e secretórias (Jak2WT 36,12%, -10,52 ±3,029 vs Jak2VF 25,60%, -17,37 ±-3,664, p = 0,0070) apresentaram menor frequência e as células NK tolerantes (Jak2WT 12,43%, 11,91 ± 2,689 vs Jak2VF 24,34%, 5,828 ± 18,00, p = 0,0016) e citotóxicas (Jak2WT 7,15%, 12,14 ± 1,670 vs Jak2VF 19,30%, 8,365 ± 15,92, p < 0,0001) apresentaram maior frequência quando expostas à leucemia do que quando expostas à medula óssea normal. Além disso, foi observada diminuição da expressão do receptor de ativação CD226 (Jak2WT 51,20%, -23,00 ±4,493 vs Jak2VF 28,20%, -33,16 ±-12,84, p = 0,0006) e aumento do receptor de inibição TIGIT (Jak2WT 14,83%, 31,27 ± 5,400 vs Jak2VF 46,10%, 19,06 ±43,48, p = 0,0003) nesta mesma comparação. Conclusões: Nossos achados demonstraram que células NK expostas às células Jak2V617F apresentaram diminuição da frequência, redução de células imaturas e aumento de células citotóxicas que estão mais ativadas quando células NK são cultivadas com soro leucêmico. Ademais, houve alterações no perfil de expressão dos receptores e ligantes, no sentido de desfavorecer a ativação e promover inibição da função NK. Sendo assim, concluímos que a exposição à doença alterou o fenótipo e a maturação funcional das células NK corroborando com a hipótese de que o meio leucêmico é capaz de levar à disfunção NK e que estratégias voltadas ao controle da relação entre ligantes e receptores funcionais NK podem ser exploradas para restaurar a imunovigilância em Neoplasias Mieloproliferativas associadas à mutação JAK2V671F.

A Mielofibrose Primária (MFP) é uma neoplasia mieloproliferativa iniciada por mutações na célula-tronco hematopoética (e. g. JAK2V617F) e pode ser classificada de acordo com o grau de fibrose na medula óssea em pré-fibrótica (MFPPF; grau de fibrose

Na Leucemia Mieloide Crônica (LMC), pacientes que alcançam resposta molecular profunda sustentada (RMP) com inibidor de tirosinoquinase (ITQ) podem ter o tratamento descontinuado. A remissão livre de tratamento (RLT) ocorre na minoria dos pacientes e não há biomarcadores preditores de manutenção ou perda de resposta. A imunidade antitumoral mediada por Linfócitos T (LT) e Natural Killer (NK) pode contribuir para manutenção e resultar em respostas distintas durante e após a descontinuação. Correlacionamos o fenótipo T e NK com a cinética de doença residual mensurável (uso de ITQ) e com a perda da RLT (descontinuação-DES). Foram incluídos 10 controles saudáveis (CT) e 23 pacientes com LMC: 8 ao diagnóstico (DX), 4 tratados por mais de 3 anos com RM menor que log 3 (RM4,5) em DES, divididos e acompanhados em meses (m), no qual, 3m (n = 13), 6m (n = 8) e 9m (n = 7), e 8 que recaíram (R) no DES. Até o ponto 6m, os pacientes estavam com 50% da dose do ITQ e, após este ponto, tiveram o ITQ suspenso. Foram avaliados frequência, subtipos e maturação de NK e LT por citometria de fluxo do sangue periférico. Os subtipos NK (CD45hiCD19−CD3−) foram definidos como secretório (CD56brightCD16−) ou citotóxico (CD56dimCD16+), e a maturação funcional das células CD19−CD3−CD56+ foi classificada: CD11b−CD27− (tolerante); CD27+CD11b− (secretória imatura); CD27+CD11b+(secretória madura) e CD11b+CD27− (citotóxica), além dos marcadores de ativação CD57 e NKp80. A frequência destas subpopulações foi avaliada em subgrupos de pacientes de acordo com o nível de RM por RT-qPCR para o gene BCR::ABL1. Em comparação aos CT, amostras de LMC em atividade (DX, RM0,05). Em relação aos subtipos de NK, DX, RM 0,05). Na maturação funcional de NK, DX, RM 0,05). Na ativação de NK, DX, RM4,5 e DES, possuem perfil mais maduro de NK, no qual a atividade citotóxica é semelhante aos CT. Entretanto, pacientes maus respondedores (RM

O monitoramento da Leucemia Mieloide Aguda (LMA) é uma estratégia de avaliação prognóstica para identificar Doença Residual Mensurável (DRM). Indica-se o estudo de DRM por métodos moleculares em casos com mutações NPM1 /translocações tipo core-binding fator; mas a citometria de fluxo multiparamétrica (CFMP) é aplicável a mais de 90% das LMAs e preditora do sucesso terapêutico. Diante da escassez no acesso a métodos moleculares em países em desenvolvimento, um protocolo de determinação de DRM na rotina laboratorial é uma necessidade não atendida. Objetivamos implantar um protocolo de DRM por CFMP para LMA e avaliá-lo quanto à sua eficiência e como fator prognóstico adicional à estratificação de risco clássica. Foram incluídos 191 pacientes com LMA de novo ou secundária à síndrome mielodisplásica, entre 18 e 65 anos (09/2015 a 02/2023), com remissão morfológica completa após tratamento com 1 ou 2 ciclos de indução (ciclo 1:3 dias de daunorrubicina 60 mg/m2 e 7 dias de citarabina 200 mg/m2; ciclo 2:3 dias de daunorrubicina 60 mg/m2 e 6 dias de citarabina 1 g/m2 2x/dia). Na Consolidação foi administrada no ciclo1:citarabina 1 g/m2 2x/dia, 6 dias e no ciclo2: idem 1 ou transplante de Medula Óssea (MO) autólogo com condicionamento BuCy. Os pacientes foram estratificados (ELN2017*-exceto mutações TP53 ou ASXL1) de acordo com cariótipo, testes moleculares e coleta de dados clínicos. As DRMs 1, 2 (Pós-indução) e 4 (Pós consolidação), foram avaliadas com: CD45, CD34, CD117, CD33, CD38, CD19, CD123, CD7, CD56, CD13, CD11b, CD14, CD64, CD300e, CD15, CD133 e HLA-DR. Utilizamos a abordagem de LAIPs e DfN, comparando dados de MO saudável e diagnóstico e sendo DRM+ a frequência de células em CD45+ ≥ 0,1% (mínimo 1 fenótipo anormal). Analisamos Sobrevida Global e Livre de Recaída (SG e SLR) e Incidência Cumulativa de Recaída (ICR) - software R (α = 5%). Não foram encontradas associações entre DRM+ e idade, leucócitos, % de blastos, subtipo morfológico e alterações citogenéticas. Observou-se 21% de DRM+ na DRM1,13% na DRM2 e 12,5 % na DRM4. Na DRM1, a frequência de DRM+ para pacientes de risco baixo, intermediário e alto foi: 46,5%, 33,4%, 20,1% e nas DRMs 2 e 4, 33,4%/ 38,9%/ 27,7% e 48,8%/ 48,8%/ 14,4%, respectivamente. Globalmente as DRMs foram definidas como LAIPs ou DfN em 72,3% e 27,7% das amostras. A expressão de CD56 foi a mais frequente (22%), seguida por alterações em CD34/CD13 (21%) e CD33 (17%). DRMsnegativas foram mais frequentes em pacientes com NPM1mut (DRM1, 69,7%) do que em FLT3-ITD (15,2%). A DRM+ após o 2ºciclo de Indução implicou em maior risco de óbito (HR = 1329 (IC;75,16-235022), p = 0,002), maior ICR (44,5% em 30 meses, p = 0,033) e menor SG (4,4 meses, p = 0,018) no risco intermediário. A detecção de DRM+ não se associou aos riscos favorável e alto. Nossos achados demonstram a padronização da DRM por CFMP em uma população brasileira com tratamento único reprodutível usando painel racional com número limitado de marcadores. A detecção de fenótipos ao diagnóstico (LAIP) é mais útil do que os fenótipos semelhantes a DfN, embora sua combinação tenha validado o tubo proposto como “consenso”, aplicável a todos os casos a despeito do fenótipo inicial. Em pacientes de risco intermediário, o resultado da DRM por CFMP se mostrou fator de risco independente para SG. Sendo este grupo correspondente à cerca 1/3 dos casos, comumente com cariótipo normal e desfechos variáveis, a identificação de fatores prognósticos adicionais e dinâmicos tem grande utilidade e pode guiar as decisões terapêuticas. Por fim, a identificação dos fenótipos de DRM definidos neste protocolo pode contribuir na busca por alvos terapêuticos para erradicação das células leucêmicas resistentes.

A Leucemia Mieloide Aguda (LMA) se origina de anormalidades genéticas ao nível das células-tronco hematopoéticas (CTH), que adquirem propriedades de células-tronco leucêmicas (CTL) com capacidade aumentada de sobrevivência e bloqueio de maturação. Hipotetizamos que a parada de maturação leucêmica por meio de definição fenotípica das células de origem possa acrescentar informações importantes sobre a biologia da LMA e contribuir para a predição de desfechos. Assim, analisamos retrospectivamente em coorte brasileira de pacientes com LMA o perfil fenotípico de maturação de blastos leucêmicos e sua associação com achados clássicos de morfologia, risco genético e informações clínicas. Foram incluídos dados de imunofenotipagem de 359 indivíduos com LMA encaminhados para o estudo ICAML2015, entre 18 e 70 anos, acompanhados durante oito anos, exceto os casos de Leucemia Promielocítica Aguda (LPA) que só foram incluídos após 2020. A expressão de CD34, CD117, CD13, CD33, CD38, (c)MPO e HLA-DR foi avaliada para a categorização nos subtipos HSC: Hematopoietic stem cell; MPP: Multipotent progenitors; CMP: Common myeloid progenitors; GMP: Granulocyte-monocyte progenitors; MP: Monocyte progenitors; GP: Granulocyte progenitors. LMAs tipo HSC foram definidas por CD34+, CD117−/+, HLA-DR+, CD13/33−, MPO−. A expressão de CD13 e 33 definiu o subtipo MPP. A expressão de cMPO entre 10-70% definiu o subtipo CMP e > 70% GMP. Os subtipos MP/GP foram diferenciados pela expressão de HLA-DR no subtipo GP. A reclassificação dos casos demonstrou a seguinte distribuição: HSC (n = 31, 8.64%); MPP (n = 77, 21.45%); CMP (n = 37, 10.31%); GMP (n = 75, 20.89%); MP (n = 50, 13.93%); GP (n = 89, 24.79%). De acordo com a estratificação de risco da European-Leukemia Net 2017, dentre os subtipos HSC, MPP, CMP e GMP predominaram o risco favorável, seguidos de intermediário e adverso. Nos subtipos MP e GP, o risco intermediário foi mais prevalente em relação ao favorável e adverso. A correlação com a classificação morfológica demonstrou que LMA sem maturação, com maturação, mielomonocítica e monocítica se distribuíram de forma equivalente nos subtipos MPP, CMP, GMP, MP e GP; no subtipo HSC não se encontrou leucemia monocítica ou promielocítica, sendo esta caracteristicamente do tipo GP e raramente GMP. Para 325 pacientes, os dados dos genes NPM1, FLT3 -ITD, BAALC, EVI1 e CEBPA estavam disponíveis, sendo encontrada com maior frequência a mutação FLT3 -ITD no subtipo MPP (26/325) e NPM1 em GP (44/325). Em algum ponto de seu seguimento 57/359 pacientes recaíram, representando nos subtipos: HSC (5/31, 16%), MPP (15/77, 19%), CMP (6/37, 16%), GMP (16/75, 21%), MP (6/50, 12%) e GP (9/89, 10%). A sobrevida global foi avaliada em 309/359 pacientes, tendo-se observado maior taxa para o subtipo GP (66%) e menor para CMP (11%) aos 24 meses de seguimento (p = 0.03). Os achados apontam que as correlações entre o estado de parada de maturação da LMA e dados clínicos de estratificação de risco e sobrevida merecem atenção e melhor exploração. É possível que a combinação dos dados genéticos com a classificação imunofenotípica de origem leucêmica seja útil para aferir prognóstico na LMA, pois é plausível que as leucemias mais maduras tenham um microambiente inflamatorio exuberante, capaz de modificar a imunidade antitumoral e contribuir para os desfechos diversos.

A Leucemia Linfoblástica Aguda (LLA) é uma neoplasia de células linfoides imaturas T ou B, sendo a segunda leucemia aguda mais comum no adulto. Acomete cerca de 1 a 5 /100.000 indivíduos por ano nos Estados Unidos e cerca de 66% dos casos são LLA-B. O tratamento da LLA-B consiste em quimio/imunoterapia de alta intensidade, com objetivo primário de restaurar a hematopoese normal. A monitorização de remissão de doença (Doença Residual Mensurável - DRM) é realizada em diferentes etapas do tratamento por citometria de fluxo multiparamétrica (CFMP), sendo a técnica eficaz na detecção de clones leucêmicos pós-tratamento com quimioterapia de alta intensidade. Para pacientes recaídos/refratários a terapia de resgate mais indicada consiste em imunoterapia com agentes específicos, chamados de terapias-alvo (ex: anti-CD3 + anti-CD19 -Blinatumomabe, anti-CD22 -Inotuzumabe + Ozogamicin), e a terapia com células T geneticamente modificadas - CAR-T (do inglês, Chimeric antigen receptor T Cell). Com o surgimento de tais tratamentos surge também a demanda por padronização da monitorização de DRM nos pacientes, visto que terapia pode resultar em perda de expressão de marcadores, o que inviabiliza a análise pelos métodos anteriormente padronizados, enfatizando a possibilidade de recaídas com clones emergentes negativos para a expressão de CD19, por exemplo. Assim, foi objetivo do presente estudo determinar a viabilidade de implantação de novo protocolo para detecção de DRM com base em seleção alternativa de antígenos de células B para pacientes submetidos à terapia convencional ou anti-CD19 por CFMP. Foram analisadas as medulas ósseas de 2 doadores saudáveis, e 2 pacientes LLA-B tratados no Hospital das Clínicas de Ribeirão Preto - FMRP-USP, sendo um submetido à terapia HyperCVAD e 1 paciente refratário, tratado com CAR-T anti-CD19. As amostras foram preparadas de acordo com protocolo Euroflow e avaliadas quanto à expressão dos marcadores (CD45, CD34, CD38, CD81, CD10, CD20, CD19, CD22, CD24, CD66b, CD66c + CD123, CD73) por CFMP em citômetro BD Canto II e analisadas em software Infinicyt por dois observadores diferentes. Os resultados da análise DRM foram expressos como a porcentagem de células nucleadas viáveis que abrigam o fenótipo anormal, sendo a positividade definida quando igual ou maior do que 0,01%. No presente estudo de viabilidade a detecção de células B foi concordante entre os diferentes tubos, independente de marcação de CD19, não havendo diferença nas frequências observadas tanto para amostras de referência como no paciente analisado (p = 0,4478, Kruskal-Wallis). Não foi observada diferença entre as frequências de células anormais detectadas pelos observadores (p = 0,4, Kruskal Wallis) e nossos resultados demonstram reprodutibilidade entre as análises realizadas, com Correlação de Pearson calculada de 0,9995 (IC: 0,9949-0,999, r2 = 0,9989). A análise de Bland-Altman demonstrou não haver divergência entre as metodologias utilizadas. Não houve diferença nas frequências obtidas dos fenótipos caracterizados como DRM+, independente da combinação de marcadores para a gate de células B (CD19xCD22, CD22xCD24, e CD24xCD66b) p = 0,667, Friedman, α=0,05). Em conclusão, nossos dados indicam que o monitoramento da DRM para pacientes com LLA-B tratados com imunoterapia anti-CD19 é possível por CFMP resultando em dados altamente concordantes. O desempenho do resultado preliminar exposto deve garantir resultados comparáveis ao serem avaliados em múltiplos centros, etapa em fase de planejamento, para garantir o avanço da padronização da resposta de DRM por CFMP.

Background: Diagnosis and treatment of acute myeloid leukemia (AML) remains difficult to this day, cure rates remain low and measurable residual disease (MRD) assessment is still challenging. Early detection of residual leukemic cells is essential to predict therapeutic success and Multiparametric Flow Cytometry (MFC) has been an effective tool for it. Here, we focus on the importance in caution when interpreting MDR in AML. Methods: Bone marrow (BM) sample was assessed to FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype (KT) and immunophenotyping. Using an 8-colour antibody panel (CD45, CD34, CD117, CD 33, HLA-DR, CD38, CD123, CD13, CD19, CD7, CD11b, CD4, CD56, CD64, CD11c, CD15, CD133, CD41a and NG2), we applied a gating strategy with fixed gates comparing normal to AML BM, settled to catch LAIPs (Leukemia Associated-Immunophenotypes), abnormal cell maturation pattern and Leukemic Stem Cell, that were detected on diagnosis and evaluated for MRD. Assays were performed at the Hematology Laboratory of the Medical School of Ribeirão Preto, University of São Paulo, in accordance to Local Ethical Boards. Results: A 21-year-old male patient with AML without maturation, diagnosed after 60 days of symptoms, presented anemia (7.9 g/dL), thrombocytopenia (26 000/μL) and white blood cell count of 5710/μL with 73% blasts in peripheral blood and 84% in BM. MFC revealed CD45dim and low side scatter; positive staining for CD117, CD34, HLA-DR, CD38, CD123, CD11c, CD64, CD15 and CD133; negative staining for CD33, CD14, CD7, CD11b, CD4, CD56, CD19, CD41a and NG2. We found no metaphases (KT), no molecular abnormality and he failed risk stratification. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day). BM was obtained at diagnosis, 30 and 33 days after 1st and 2nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. The positivity of a specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved at diagnosis and follow-ups: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122%. Complete remission was achieved by day 30 after 1st induction and no change in the outcome was reported. Absent CD33 was observed in diagnosis and follow-ups, during and after treatment. Discussion: Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a common myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33low blasts are associated to a more mature AML and its high expression is related to adverse landscapes, highlighting the importance of CD33 evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse or poor outcomes. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with absent CD33 maintained from diagnosis to MRD follow-ups, mimicking MRD positivity. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results interpretation, as well as constant training of the team.

Background: Acute myeloid leukemia with monocytic blast differentiation (AML-MC) is a rare malignancy (5-10% incidence) with a challenging diagnosis, classified as “non otherwise specified” AML (AML NOS), unless recurrent genetic abnormalities, therapy-related or associated myelodysplasia-related changes are reported (WHO 2017). We observed a high incidence of AML-MC and aimed to assess its molecular and immunophenotypic features in a multicenter study in Brazil. Methods: 387 AML bone marrow samples (18-84 years old) from 10 Brazilian centers enrolled in International Consortium of Acute Leukemias (ICAL) Study – IC-AML2015, is in accordance Local Ethical Boards committee. From which, 149 were morphologically diagnosed with AML-MC. FLT3 and NPM1 mutations, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and immunophenotype were assessed. Mononuclear cells were stained with CD45, CD34, CD117, CD11c, CD64 and CD15. Cell acquisition/analysis was performed in FACS Canto II /FACS Diva software (BD Bioscience). Positivity was considered when ≥20% of the leukemic cells. Treatment consisted on 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day) and/or BM transplantation. Results: AML-MC corresponded to 38.50% of cases enrolled in the study. Stratifying according to ELN2017 (without TP53 and ASXL1), 23% were Favorable, 22% Intermediate and 19% Adverse risk. 35% of patients failed Stratification or had Not Assessed Information. Molecular Assessment showed positive detections for FLT3 (17.48%), RUNX1-RUNX1T1 (2.80%), CBFB-MYH11 (8.51%) and NPM1 (23.77%). According to treatment response evalutation, complete remission CR was achieved by 49,14%, 55,8%, 53,70% and 59,46% of patients on 1st,2ndInductionof remission cycle of chemotherapy, 1st and 2sn cycle of Consolidaton of chemotherapy, and 26.37% of patients relapsed. Multiparametric Flow Cytometry (all risks) showed 39% CD34−, 61% CD34+,11% CD34−/CD117−, 13% CD117, 87% CD117+, 95% CD33+, 89% CD38+, 47% CD123+, 8% CD14+, 21% CD7+, 44% CD11b+, 19% CD56+, 29% CD13−, 2% CD19+, 27% CD64+, 74% CD11c+ 27%, CD64+CD11c+, 19% CD15+/CD133+ and 60% CD15+ cells. Among AML-NOS patients, 26% presented CD15+ and none were stratified as Favorable risk (6% Intermediate, 17% Adverse and 4% Failed Stratification). Discussion: Since AML-MC is known to be rare, accounting for 5-10% of AML cases worldwide, its high incidence in our study seemed to be odd and raised curiosity in the molecular and phenotypic context. The fact that a great number of these cases are intermediate ELN2017 risk brings up the necessity to look at them closer, since this is a challenging group when it comes to therapy decisions. Although good response to treatment was noticed, immunophenotypically CD15 expression without molecular abnormalities was more frequent in intermediate and adverse risk. These findings may direct that high rates of good treatment response and favorable outcome in these patients are more likely to be due to favorable molecular abnormalities, while expression of CD15 alone may be associated with a more aggressive disease. Knowing that, it should be of value to investigate whether CD15 could be a good prognostic marker.

Objetivos: O substrato do receptor de insulina 2 (IRS2) interage com receptores envolvidos no processo hematopoético e sua expressão é reduzida em pacientes com síndrome mielodisplásica. Em trabalho anterior, nosso grupo descreveu os resultados da avaliação da reconstituição da hematopoese a curto prazo após transplante de medula óssea (TMO) de camundongos nocaute para Irs2 (Irs2 KO) em receptores C57BL/6. A análise, 4 e 8 semanas após o transplante, evidenciou uma redução nos níveis de hemácias, hematócrito e hemoglobina no grupo transplantado com células de medula óssea de animais Irs2 KO em comparação com animais receptores de camundongos selvagem (WT). Após 8 semanas do transplante, a contagem de plaquetas também reduziu em animais receptores de Irs2 KO. O presente trabalho tem como objetivo principal, avaliar os efeitos em longo prazo do TMO de células Irs2 KO sobre a constituição das populações hematopoéticas. Material e métodos: A hematopoese de camundongos C57BL/6 previamente submetidos a TMO com células de animais B6.129-Irs2tm1Mfw (WT: n = 3 e Irs2 KO: n = 5) foi avaliada 12 meses pós-TMO. Os parâmetros hematológicos do sangue periférico (SP) foram avaliados por hemograma automatizado. O quimerismo e a constituição das populações hematopoéticas do SP, medula óssea (MO) e baço foi analisada por citometria de fluxo. Resultados: Os níveis de quimerismo se mantiveram elevados após 12 meses de transplante, variando entre 89,1 a 98,1% de células CD45.2 no sangue periférico. O quimerismo não diferiu entre os grupos, com mediana de 96,1 e 93,6 para os receptores de animais WT e Irs2 KO, respectivamente. Não houve diferença significativa entre o peso corporal e peso do baço entre os grupos (todos p > 0,05). A avaliação dos parâmetros hematológicos por hemograma automatizado revelou uma redução de glóbulos brancos (WT: 13,7 x 103/μL vs. Irs2 KO: 8,7 x 103 μL) e plaquetas (WT: 1226 x 103/μL vs. Irs2 KO: 266 x 103/μL) no grupo que recebeu células Irs2 KO; todos p < 0,05. Não houve diferença na série vermelha, incluindo glóbulos vermelhos, hemoglobina e hematócrito entre os grupos. Camundongos transplantados com células Irs2 KO apresentaram aumento da frequência de células-tronco (CTHs), incluindo a população LSK (Irs2 KO: 1,08% vs. WT: 0,33%); LT-HSC (Irs2 KO: 0,4% vs. WT: 0,08%) e ST-HSC (Irs2 KO: 0,14 % vs. WT:0,05%); todos p < 0,05. A frequência de progenitores mieloides foi superior em animais que receberam células de Irs2 KO (p = 0,03). Entretanto, o aumento das frações de progenitor mieloide comum (CMP), progenitor megacariocítico-eritrocítico (MEP) e progenitor granulocítico-macrofágico (GMP) não atingiu significância estatística (todos p > 0,05). Receptores de Irs2 KO apresentaram um aumento na subpopulação EriB [Irs2 KO: 22,1% vs. WT:15,9%). As populações de linfócitos B, linfócitos T e células mieloides não diferiram entre os grupos (todos p > 0,05). Discussão e conclusão: A avaliação final dos animais receptores indicou que o grupo que recebeu células Irs2 KO apresentou redução de leucócitos e plaquetas. Aos 12 meses, os animais não apresentaram redução da série vermelha, diferindo dos resultados obtidos nas avaliações em curto prazo. As populações de progenitores mieloides e de CTHs, incluindo LT-HSC e ST-HSC estavam aumentadas em animais que receberam células de doadores nocaute para Irs2. Os achados sugerem uma alteração funcional das CTHs, levando a um aumento compensatório dessas populações. Financiamento: FAPESP, CAPES e CNPq.

Myeloproliferative neoplasms (MPN) belong to a group of clonal diseases of hematopoietic stem cells characterized by aberrant proliferation of mature myeloid lineages. The constitutive activation of the JAK2/STAT signaling pathway is now well established to play a central role in MPN pathogenesis; however, accumulating evidence now indicates that the IGF1R-mediated signaling pathway contributes to the maintenance of the malignant phenotype. Studies using inhibitors of IGF1-mediated signaling have reported cytotoxic effects in cellular and murine models of MPN, but no consensus has been reached regarding the potency and efficacy of inhibitors of the IGF1R-related pathway in this context. In the present study, we compared the potency and efficacy of three inhibitors of IGF1R-related pathways in a JAK2 V617F -driven cellular model. These inhibitors (NT157, OSI-906, and NVP-AEW54) present antineoplastic activity with similar efficacy in Ba/F3 JAK2 V617F cells, with NT157 showing the greatest potency. Both the induction of apoptosis and reduction in cell proliferation were associated with the observed reduction in cell viability. Downregulation of JAK2/STAT signaling was an advantageous off-target effect of all three inhibitors. These preclinical studies reinforce the potential of the IGF1R-related pathway as a therapeutic target in MPN., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Introduction: Telomere length (TL) is a biomarker of cellular proliferative history. In healthy individuals, leukocyte TL shortens with age and associates with the lifespan of men and women. However, most of studies had used linear regression models to address the association of the TL attrition, aging and sex., Methods: We evaluated the association between the TL, aging and sex in a cohort of 180 healthy subjects by quantile regression. The TL of nucleated blood cells was measured by fluorescent in situ hypridization (flow-FISH) in a cohort of 89 men, 81 women, and 10 umbilical cord samples. The results were validated by quantitative polymerase chain reaction (qPCR) and compared to a linear regression analysis., Results: By quantile regression, telomere dynamics slightly differed between sexes with aging: women had longer telomeres at birth and slower attrition rate than men until the sixth decade of life; after that, TL eroded faster and became shorter than that in men. These differences were not observed by linear regression analysis, as the overall telomere attrition rates in women and men were similar (42 pb per year, p < 0.0001 vs. 45 pb kb per year, p < 0.0001). Also, qPCR did not recapitulate flow-FISH findings, as the telomere dynamics by qPCR followed a linear model., Conclusion: The quantile regression analysis accurately reproduced a third-order polynomial TL attrition rate in both women and men, but it depended on the technique applied to measure TL. The Flow-FISH reproduced the expected telomere dynamics through life and, differently from the qPCR, was able to detect the subtle TL variations associated with sex and aging., (Copyright © 2021 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2 V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients., Aims: We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2 V617F -knockin MPN mice., Results: In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2 V67F cells. Long-term treatment with 40 mg/kg phenformin in Jak2 V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2 V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice., Conclusions: Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2 V617F -knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Stathmin 1 (STMN1) is a microtubule-destabilizing protein highly expressed in hematological malignancies and involved in proliferation and differentiation. Although a previous study found that the PML-RARα fusion protein, which contributes to the pathophysiology of acute promyelocytic leukemia (APL), positively regulates STMN1 at the transcription and protein activity levels, little is known about the role of STMN1 in APL. In this study, we aimed to investigate the STMN1 expression levels and their associations with laboratory, clinical, and genomic data in APL patients. We also assessed the dynamics of STMN1 expression during myeloid cell differentiation and cell cycle progression, and the cellular effects of STMN1 silencing and pharmacological effects of microtubule-stabilizing drugs on APL cells. We found that STMN1 transcripts were significantly increased in samples from APL patients compared with those of healthy donors (all p < 0.05). However, this had no effect on clinical outcomes. STMN1 expression was associated with proliferation- and metabolism-related gene signatures in APL. Our data confirmed that STMN1 was highly expressed in early hematopoietic progenitors and reduced during cell differentiation, including the ATRA-induced granulocytic differentiation model. STMN1 phosphorylation was predominant in a pool of mitosis-enriched APL cells. In NB4 and NB4-R2 cells, STMN1 knockdown decreased autonomous cell growth (all p < 0.05) but did not impact ATRA-induced apoptosis and differentiation. Finally, treatment with paclitaxel (as a single agent or combined with ATRA) induced microtubule stabilization, resulting in mitotic catastrophe with repercussions for cell viability, even in ATRA-resistant APL cells. This study provides new insights into the STMN1 functions and microtubule dynamics in APL., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34 + samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro . Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34 + samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro , treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo , the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Almeida, Pereira-Martins, Weinhäuser, Ortiz, Cândido, Lange, De Abreu, Mendonza, de Deus Wagatsuma, Do Nascimento, Paiva, Alves-Paiva, Bonaldo, Nascimento, Alves-Filho, Scheucher, Lima, Schuringa, Ammantuna, Ottone, Noguera, Araujo and Rego.)

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1 T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1 T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

Eltrombopag has been added to first-line treatment of immune aplastic anaemia (AA), resulting in higher responses. We analysed marrow samples of AA patients who responded to immunosuppressive therapy (IST) alone or in combination with eltrombopag for the composition of the haematopoietic stem and progenitor cell (HSPC) compartment. The number of CD34 + cells and multipotent progenitors was higher in patients treated with eltrombopag (P < 0·005; P < 0·05; respectively), but not the number of stem cells. No aberrant phenotype was observed. These results indicate that eltrombopag augments CD34 + cells in vivo and preferentially expands multipotent progenitors, but not stem cells., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Background: Differentiation syndrome (DS) is the main life-threatening adverse event that occurs in acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (ATRA). Cytokine imbalances have been reported to play role during the developing of acute promyelocytic leukemia differentiation syndrome (APL-DS). However, the relationship between the plasma cytokine levels and their prognostic value for the prediction of DS developing in patients with APL during the treatment with ATRA and anthracyclines has not been previously reported., Methods: In this study, we followed an APL cohort (n = 17) over 7 days of ATRA therapy in DS (n = 6) and non-DS groups (n = 11). Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α were measured in the peripheral blood plasma from 17 patients with APL and 11 healthy adult controls by using the cytometric bead array method., Results: In non-DS patients, IL-8 plasma levels were significantly reduced in the seventh day of ATRA treatment (34.16; 6.99 to 147.11 pg mL - 1 in D0 vs. 10.9; 0 to 26.81 pg mL - 1 in D7; p = 0.02) whereas their levels did not discriminate between DS and non-DS development during the entire induction period (all p > 0.05 in D0, D3, and D7). No significant differences were found in IL-6 levels between groups (p > 0.05 in D0-D7). Other cytokines tested were all undetectable in patients with APL or healthy controls., Conclusions: We demonstrated that the modulation of IL-8 following ATRA treatment may occur regardless of the development of DS and, therefore, does not appear to be a predictive biomarker to monitor the APL-DS.

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.

JAK2 V617F can mimic growth factor signaling, leading to PI3K/AKT/mTOR activation and inhibition of autophagy. We hypothesized that selective inhibition of JAK1/2 by ruxolitinib could induce autophagy and limit drug efficacy in myeloproliferative neoplasms (MPN). Therefore, we investigated the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, to determine the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2 V617F cells, and to verify the frequency and clinical impact of autophagy-related gene mutations in patients with MPNs. In SET2 JAK2 V617F cells, ruxolitinib treatment induced autophagy and modulated 26 out of 79 autophagy-related genes. Ruxolitinib treatment reduced the expressions of important autophagy regulators, including mTOR/p70S6K/4EBP1 and the STAT/BCL2 axis, in a dose- and time-dependent manner. Pharmacological inhibition of autophagy was able to significantly suppress ruxolitinib-induced autophagy and increased ruxolitinib-induced apoptosis. Mutations in autophagy-related genes were found in 15.5% of MPN patients and were associated with increased age and a trend towards worse survival. In conclusion, ruxolitinib induces autophagy in JAK2 V617F cells, potentially by modulation of mTOR-, STAT- and BCL2-mediated signaling. This may lead to inhibition of apoptosis. Our results suggest that the combination of ruxolitinib with pharmacological inhibitors of autophagy, such as chloroquine, may be a promising strategy to treat patients with JAK2 V617F -mutated MPNs.

Recent data indicate that IGF1R/IRS signaling is a potential therapeutic target in BCR-ABL1-negative myeloproliferative neoplasms (MPN); in this pathway, IRS2 is involved in the malignant transformation induced by JAK2 V617F , and upregulation of IGF1R signaling induces the MPN phenotype. NT157, a synthetic compound designed as an IGF1R-IRS1/2 inhibitor, has been shown to induce antineoplastic effects in solid tumors. Herein, we aimed to characterize the molecular and cellular effects of NT157 in JAK2 V617F -positive MPN cell lines (HEL and SET2) and primary patient hematopoietic cells. In JAK2 V617F cell lines, NT157 decreased cell viability, clonogenicity, and cell proliferation, resulting in increases in apoptosis and cell cycle arrest in the G 2 /M phase (p < 0.05). NT157 treatment inhibited IRS1/2, JAK2/STAT, and NFκB signaling, and it activated the AP-1 complex, downregulated four oncogenes (CCND1, MYB, WT1, and NFKB1), and upregulated three apoptotic-related genes (CDKN1A, FOS, and JUN) (p < 0.05). NT157 induced genotoxic stress in a JAK2/STAT-independent manner. NT157 inhibited erythropoietin-independent colony formation in cells from polycythemia vera patients (p < 0.05). These findings further elucidate the mechanism of NT157 action in a MPN context and suggest that targeting IRS1/2 proteins may represent a promising therapeutic strategy for MPN.

Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold ( n  = 8) and decreased tumor weight by 2.7-fold ( n  = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.

The IGF1R/IRS1 signaling is activated in acute lymphoblastic leukemia (ALL) and can be targeted by the pharmacological inhibitors NT157 (IGF1R-IRS1/2 inhibitor) and OSI-906 (IGF1R/IR inhibitor). Here we investigate the cellular and molecular effects of NT157 and OSI-906 in ALL cells. NT157 and OSI-906 treatment reduced viability, proliferation and cell cycle progression in ALL cell lines. Similarly, in primary samples of patients with ALL, both OSI-906 and NT157 reduced viability, but only NT157 induced apoptosis. NT157 and OSI-906 did not show cytotoxicity in primary samples from healthy donor. NT157 and OSI-906 significantly decreased Jurkat cell migration, but did not modulate Namalwa migration. Consistent with the more potent effect of NT157 on cells, NT157 significantly modulated expression of 25 genes related to the MAPK signaling pathway in Jurkat cells, including oncogenes and tumor suppressor genes. Both compounds inhibited mTOR and p70S6K activity, but only NT157 inhibited AKT and 4-EBP1 activation. In summary, in ALL cells, NT157 has cytotoxic activity, whereas OSI-906 is cytostatic. NT157 has a stronger effect on ALL cells, and thus the direct inhibition of IRS1 may be a potential therapeutic target in ALL., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.

Introduction: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year., Methods: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR)., Results: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods., Conclusions: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

The recurrent gain-of-function JAK2 V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor ruxolitinib indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that exerts selective antineoplastic activity in hematological malignancies. In the present study, we investigate and compare effects of metformin and ruxolitinib alone and in combination on cell signaling and cellular functions in JAK2 V617F -positive cells. In JAK2 V617F -expressing cell lines, metformin treatment significantly reduced cell viability, cell proliferation, clonogenicity, and cellular oxygen consumption and delayed cell cycle progression. Metformin reduced cyclin D1 expression and RB, STAT3, STAT5, ERK1/2 and p70S6K phosphorylation. Metformin plus ruxolitinib demonstrated more intense reduction of cell viability and induction of apoptosis compared to monotherapy. Notably, metformin reduced Ba/F3 JAK2 V617F tumor burden and splenomegaly in Jak2 V617F knock-in-induced MPN mice and spontaneous erythroid colony formation in primary cells from polycythemia vera patients. In conclusion, metformin exerts multitarget antileukemia activity in MPN: downregulation of JAK2/STAT signaling and mitochondrial activity. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action and provides insights for development of alternative/complementary therapeutic strategies for MPN.

Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by abnormal proliferation and accumulation of lymphoblasts in the hematopoietic system. Stathmin 1 is a proliferation marker for normal lymphocytes, which has been described as highly expressed in ALL patients and functionally important for leukemia phenotype. In the present study, we expand our previous observations and aim to investigate Stathmin 1 expression and its impact on laboratory features and clinical outcomes in an independent cohort of ALL patients, and to verify the effects of paclitaxel treatment on Stathmin 1 phosphorylation and cell viability in ALL cell lines. In ALL patients, Stathmin 1 expression was significantly increased, associated with lower age onset and positively correlated with white blood cell counts, but did not impact on clinical outcomes. Functional assays revealed that paclitaxel induces Stathmin 1 phosphorylation at serine 16 (an inhibitory site), microtubule stability and apoptosis in Jurkat and Namalwa cell lines. Paclitaxel treatment did not modulate cell viability of normal peripheral blood leukocytes. In conclusion, our data confirm increased levels of Stathmin 1 in ALL patients and that therapeutic doses of paclitaxel inhibits Stathmin 1 function and promote microtubule stability and apoptosis in ALL cells.

Background: Evidence suggests that monoclonal B-cell lymphocytosis precedes all chronic lymphocytic leukemia cases, although the molecular mechanisms responsible for disease progression are not understood. Aberrant miRNA expression may contribute to the pathogenesis of chronic lymphocytic leukemia. The objective of this study was to compare miRNA expression profiles of patients with Binet A chronic lymphocytic leukemia with those of subjects with high-count monoclonal B-cell lymphocytosis and healthy volunteers (controls)., Methods: Twenty-one chronic lymphocytic leukemia patients, 12 subjects with monoclonal B-cell lymphocytosis and ten healthy volunteers were enrolled in this study. Flow cytometry CD19 + CD5 + -based cell sorting was performed for the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups and CD19 + cells were sorted to analyze the control group. The expressions of miRNAs (miR-15a, miR-16-1, miR-29b, miR-34a, miR-181a, miR-181b and miR-155) were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)., Results: Significant differences between the expressions in the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups were restricted to the expression of miR-155, which was higher in the former group. A comparison between healthy controls and monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia patients revealed higher miR-155 and miR-34a levels and lower miR-15a, miR-16-1, miR-181a and miR-181b in the latter group., Conclusions: Our results show a progressive increase of miR-155 expression from controls to monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. The role of miR-155 in the development of overt chronic lymphocytic leukemia in individuals with monoclonal B-cell lymphocytosis must be further analyzed., (Copyright © 2017 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.)

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic clinical entity characterized by the proliferation of monoclonal B cells not meeting the diagnosis criteria for chronic lymphocytic leukemia (CLL). MBL may precede the development of CLL, but the molecular mechanisms responsible for disease progression and evolution are not completely known. Telomeres are usually short in CLL and their attrition may contribute to disease evolution. Here, we determined the telomere lengths of CD5+CD19+ cells in MBL, CLL, and healthy volunteers. Twenty-one CLL patients, 11 subjects with high-count MBL, and 6 with low-count MBL were enrolled. Two hundred and sixty-one healthy volunteers aged 0 to 88 years were studied as controls. After diagnosis confirmation, a flow cytometry CD19+CD5+-based cell sorting was performed for the study groups. Telomere length was determined by qPCR. Telomere length was similar in the 3 study groups but shorter in these groups compared to normal age-matched subjects that had been enrolled in a previous study from our group. These findings suggest that telomere shortening is an early event in CLL leukemogenesis.

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by presence of the oncoprotein BCR-ABL1, which have constitutive tyrosine kinase activity. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Acquisition of resistance to tyrosine kinase inhibitors has emerged as a problem for CML patients and the identification of novel targets with an important contribution for CML phenotype is of interest. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1+ K562 cells. Our results indicate that reversine reduces AURKA and AURKB expression, leads to reduction of cell viability and increased apoptosis in a dose- and time-dependent manner, as well as, induces mitotic catastrophe in K562 cells. Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anticancer opportunities for CML., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2) = 0.60; p<0.0001) and patients (R(2) = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2) = 0.35; p<0.0001) and low correlation for patients (R(2) = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2) = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R(2) = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8 ± 7.1%) and qPCR (9.5 ± 7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.

Background: CD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome., Methods: The gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples., Results: miR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate., Conclusions: miR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

The vitamin E derivative (+)α-tocopheryl succinate (α-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARα transgenic mice, we demonstrated that α-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that α-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, α-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for α-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition., (2010 Elsevier B.V. All rights reserved.)

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58muM, for NB4 and NB4-R2, respectively. alpha-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis.

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor alpha (RARalpha) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARalpha fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARalpha develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARalpha TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 +/- 16.68, 10.83 +/- 8.11, 7.4 +/- 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARalpha TM present a specific immunophenotype.