Research progress of FLT3 inhibitors and drug resistance mechanisms in acute myeloid leukemia / 中国临床药理学与治疗学

The FMS-like tyrosine kinase 3 (FLT3) gene mutation is the most common genetic mutation in acute myeloid leukemia (AML) and is associated with poor prognosis. Various targeted inhibitors have been developed for FLT3 mutations and have shown promising clinical efficacy. However, the emergence of resistance poses new challenges for targeted therapy in AML. This article provides an overview of the pathological and prognostic role of FLT3 mutations in AML, the current research progress on commonly used FLT3 inhibitors (type I and type II), the mechanisms of FLT3 inhibitor resistance, and strategies for overcoming resistance.

GRK2 inhibits Flt-1+ macrophage infiltration and its proangiogenic properties in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/- mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/- mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.

Incidence and clinical significance of FLT3 and nucleophosmin mutation in childhood acute myeloid leukemia in Chile

Abstract Introduction Acute myeloid leukemia (AML) is a heterogeneous disease and approximately one-third of its carriers do not have evident genetic abnormalities. The mutation of specific molecular markers, such as fms-like tyrosine kinase 3 (FTL3) internal tandem duplication (ITD), FLT3 tyrosine kinase domain (TKD) and nucleophosmin (NPM1), are associated with an adverse and favorable prognosis, respectively. Objective The objective was to determine the prevalence of FLT3/ITD and NPM1 in Chilean patients and their association with clinical data and prognosis. Method and Results Two hundred and thirty-two children were studied between 2011 and 2017, the median being 8.6 years (ranging from 1 to 18 months). Acute promyelocytic leukemia (APL) was diagnosed in 29%. The FLT3/ITD-mutated in non-promyelocytic AML was at 10% (14/133) and the FLT3/TKD, at 3.7% (2/54). In APL, it was at 25.4% (16/63). In non-promyelocytic AML, the FLT3/ITD-mutated was associated with a high leucocyte count, the median being 28.5 x mm3 (n= 14) versus 19.4 x mm3 (n= 119), (p= 0.25), in non-mutated cases. In APL, the median was 33.6 x mm3 (n= 15) versus 2.8 x mm3 (n= 47), (p < 0.001). The five-year overall survival (OS) in non-promyelocytic AML with non-mutated and mutated FLT3/ITD were 62.7% and 21.4%, respectively, (p < 0.001); the 5-year event-free survival (EFS) were 79.5% and 50%, respectively, (p < 0.01). The five-year OS in APL with non-mutated and mutated FLT3/ITD was 84.7% and 62.5%, respectively, (p= 0.05); the 5-year EFS was 84.7% and 68.8%, respectively, (p= 0.122). The NPM1 mutation was observed in 3.2% (5/155), all non-promyelocytic AML with the normal karyotype. Conclusion The FLT3/ITD mutation was observed more frequently in APL and associated with a higher white cell count at diagnosis. However, the most important finding was that the FLT3/ITD mutation was associated with a shorter survival in non-promyelocytic AML.

Research progress of anti-acute marrow leukemia drugs by targeting FLT3 / 药学学报

Acute myeloid leukemia (AML) is a genetic heterogeneous disease in which primordial and juvenile myeloid cells proliferate or accumulate abnormally in bone marrow, peripheral blood and other tissues, resulting in damage to normal hematopoietic function. Studies have shown that about 30% of AML patients have FMS-like tyrosine kinase 3 (FLT3), FLT3 abnormal regulation is closely related to the occurrence and development of AML. At present, FLT3 has become an important target for developing small molecular targeted drugs. Currently, a variety of FLT3 inhibitors and FLT3 degraders have been developed targeting FLT3, and some compounds have exhibited good anti-AML activity. This article summarizes and sorts out the current mainstream drugs for AML therapeutic targeting FLT3, in order to provide a reference for the development and design of AML drugs.

Development and application of a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain of acute myeloid leukemia / 中国生物制品学杂志

@#ObjectiveTo develop a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain(FLT3-TKD)of acute myeloid leukemia(AML)and apply to the monitor of minimal residual disease(MRD).MethodsRecombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed respectively and mixed at certain ratios.The obtained standard plasmids with mutation rates of 50%,1%,0.1% and 0% respectively were determined by restriction fragment length polymorphism(RFLP)in combination with Sanger method.The plasmid DNA standards and blood DNA standards,at various FLT3-D835Y mutation rates,were determined by the developed method to verify the sensitivity.The genomic DNA samples of patients with AML before and after treatment were determined by the developed method to monitor the MRD.ResultsSequencing proved that both the recombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed correctly.The sensitivity of developed method increased to 0.1% through Sanger method combined with digestion with EcoR Ⅴ/Xho Ⅰ and recovery of mutant fragments in determination of purified plasmid DNA and collected blood DNA samples.MRD was detected in the peripheral blood sample of a patients with AML in complete remission period by the developed method but not by Sanger method.ConclusionA highly sensitive method for detection of FLT3-TKD mutation was developed,which was of an important clinical significance in guiding the treatment of AML and monitoring the MRD in complete remission period.

Analysis of laboratory characteristics and evaluation of prognostic value of patients with NPM1 mutated acute myeloid leukemia / 中华检验医学杂志

Objective:To analyze the clinical and laboratory characteristics of acute myeloid leukemia (AML) patients with NPM1 mutation, and to explore the prognostic factors.Methods:A total of 77 AML patients with NPM1 gene mutation admitted to Hebei Yanda Ludaopei Hospital from May 1st 2012 to December 31st 2021 were enrolled in the study, including 34 male and 43 female patients. The median age was 40 (3, 68) years old. Patients were selected and divided into 4 groups according to the morphological FAB classification. There were 29 cases (37.7%) of M1 type, 13 cases (16.9%) of M2 type, 23 cases (29.9%) of M4 type, and 12 cases (15.5%) of M5 type. The clinical characteristics, bone marrow/peripheral blood cell morphology, immunophenotype, cytogenetics, molecular biology and overall survival of different groups were retrospectively analyzed, and the risk factors affecting the prognosis of AML were also explored. Cox multivariate regression was used to analyze the clinical influencing factors of survival and prognosis.Results:The white blood cell counts were highest in M4 and M5 patients and lowest in M2 patients, while no significant difference in the red blood cell, hemoglobin, and platelet counts( P>0.05). Morphologically, there were significant differences in the percentage of blasts and blasts with cup-like nuclei on bone marrow (BM) and peripheral blood (PB). The proportion of blasts in BM and PB was the highest in M1 and the lowest in M2 ( P<0.001). The positive rate of blasts with cup-like nuclei was the highest in M1 and the lowest in M5 of BM ( P<0.001), while the highest in M2 and the lowest in M5 of PB ( P=0.006). The scores of myeloperoxidase and chloroacetate esterase were all the highest in M1 and the lowest in M5 ( P<0.001, 0.001, respectively). In terms of molecular biology, the occurence rate of blasts combined with DNMT3A mutation was the highest in M4 and the lowest in M2 ( P=0.044), while those combined with FLT3-ITD mutation was the highest in M4 and the lowest in M5 ( P=0.002). In immunophenotype, there were significant differences in the expression positivities of seven antigens including HLA-DR, CD56, CD11c, CD15, CD14, CD96 and cMPO ( P<0.05). Multivariate COX regression analysis showed that no recurrence after treatment ( P<0.001), complete remission after treatment ( P=0.015) and transplantation ( P<0.001) were correlated with overall survival (OS). No recurrence after treatment ( P=0.033), transplantation ( P=0.027), no mutation of FLT3-ITD ( P=0.040), and hemoglobin concentration ( P=0.023) were associated with relapse-free survival (RFS). Survival analysis by Kaplan-Meier curve showed that there was no significant difference in survival time between the M1, M2, M4 and M5 groups in OS and RFS. Conclusion:There were significant differences in the white blood count, the percentage of blasts and blasts with cup-like nuclear morphology, cytochemical staining (MPO integration, CE integration and percentage of NAS-DCE), gene mutation (DNMT3A and FLT3-ITD) and immunophenotypes (HLA-DR, CD56, CD11c, CD15, CD14, CD96 and cMPO) between the four groups. The multivariate analysis revealed that no recurrence after treatment and transplantation were independent prognostic factors in NPM1 mut AML patients. On the other hand, FLT3-ITD mutation and hemoglobin concentration were associated with RFS and complete remission after treatment was associated with OS in the entire NPM1 mut cohort.

Estudio preclínico en primate no-humano con [18F]Fluorotimidina. dosimetría y biodistribución / [18F]Fluorothymidine preclinical study in non-human primate. Dosimetry and biodistribution

Resumen En el linfoma Hodgkin, la tomografía por emisión de positrones (PET-TC) forma parte de los nuevos algoritmos diagnósticos y de valoración de respuesta al tratamiento como método eficaz para evaluar supervivencia y pronóstico de la enfermedad, ya sea a través del PET-TC interino con 2-[18F]fluoro- 2-desoxi-D-glucosa, ([18F]FDG), como también del PET-TC al final de la terapéutica. Sin embargo, la [18F]FDG presenta una baja especificidad en linfoma no Hodgkin de grandes células B. Ante la aprobación en nuestro país del radiotrazador 3´-desoxi-3´-[18F]fluorotimidina, [18F]FLT, indicador de proliferación celular de fase S, éste resultaría un prometedor radiofármaco de uso diagnóstico frente a [18F]FDG. Por lo tanto, el objetivo de este estudio fue valorar la utilización de [18F]FLT mediante un modelo animal en primates no humanos Sapajus cay. Se obtuvieron imágenes de cuerpo entero para evaluar la biodistribución y realizar un cálculo dosimétrico en la médula ósea, dado que este es un órgano crítico por la permanencia del radiofármaco. Para órganos de inte rés, se trazaron curvas de actividad en función del tiempo y se calculó la actividad acumulada normalizada. La dosis media absorbida en la médula ósea se determinó aplicando el esquema conocido como Medical Internal Radiation Dosimetry (MIRD). La dosis media obtenida en el modelo animal por unidad de actividad administrada fue de 8.7 μGy/MBq. Este resultado se extrapoló a un modelo humano adulto resultando en 32 μGy/MBq, de lo que se desprende que PET-TC con [18F]FLT es una herramienta segura para uso diagnóstico y de seguimiento en pacientes con enfermedad oncológica linfoproliferativa u otros tumores sólidos.

Abstract Positron emission tomography-computed tomography (PET-CT) is part of the new diagnostic and therapeutic algorithms for Hodgkin lymphoma. PET-CT is a valuable tool for the assessment of treatment response and prognosis, both by means of interim PET-CT with 2-[ 18F]fluoro-2-deoxy-D-glucose ([18F]FDG) as well as end of treatment (EOT) PET-CT. Given the low specificity of [ 18F]FDG for the diffuse large B cell lymphoma (DLBCL), there is an emerging need for a more specific radiopharmaceutical agent. The recent approval of the radiotracer 3´-deoxy-[18F]-3´-flourothymidine ([18F]FLT), a phase-S mitosis cell proliferation marker, for clinical application in our country, shows as a promising radiopharmaceutical for diagnostic use with incremental value over [18F]FDG. In this study, non-human primates (Sapajus cay) were studied. PET-CT study was performed after the injection of [18F]FLT. Whole-body images were obtained to evaluate the biodistribution and to calculate the dosimetry of bone marrow, as this is a critic organ to this radiotracer. Time-activity curves were traced, normalized activity uptake of the organs of interest were calculated, and mean absorbed dose was also calculated using the established Medical Internal Radiation Dosimetry (MIRD) scheme. The mean dose obtained in the animal model per unit of activity administered was 8.7 μGy/MBq. This result was extrapolated to an adult human model resulting in 32 μGy/MBq, thereby suggesting that [18F]FLT is a secure diagnostic tool to be used on the tracing of patients with DLBCL.

Biomarcador FLT3-ITD: perspectivas y retos / FLT3-ITD biomarker: perspectives and challenges

Introducción: Las mutaciones del gen que codifica para el factor de la tirosina quinasa 3 (FLT3) son de especial importancia en la leucemia mieloide aguda debido a que sirven de guía para la confirmación del diagnóstico, la estimación del pronóstico y la toma de decisiones terapéuticas. Entre las alteraciones más importantes está la duplicación interna en tándem (FLT3-ITD). Objetivo: Exponer los aspectos más relevantes respecto al biomarcador FLT3-ITD en el contexto de la leucemia mieloide aguda. Métodos: Se realizó una búsqueda de artículos científicos actualizados en los idiomas inglés y español, en PubMed, EMBASE, Google Scholar y SciELO. Se seleccionaron artículos publicados en los últimos cinco años. Se revisaron los aspectos más relevantes sobre el biomarcador en el contexto de la leucemia mieloide aguda, su base biológica, el impacto del tamaño de los fragmentos y la carga alélica en la estimación del pronóstico de los pacientes, las nuevas estrategias terapéuticas y los retos en cuanto a los métodos de laboratorio para su diagnóstico. Análisis y síntesis de la información: Más allá de la positividad o no de dicho biomarcador, el tamaño de la duplicación interna en tándem, así como la carga alélica -determinada por la razón alelo mutado/alelo salvaje-, podrían tener un gran impacto en el pronóstico. Sin embargo, persisten diferencias en los criterios para establecer los algoritmos de predicción del riesgo, el punto de corte a utilizar como referencia y el protocolo de laboratorio específico para un estudio más detallado del biomarcador. Conclusiones: La comunidad científica necesita seguir trabajando en el esclarecimiento de la utilidad práctica de estos parámetros, validándolos en series amplias y diversas epidemiológicamente. Se debe determinar el punto de corte exacto para comparar la razón y estandarizar los métodos de laboratorio más adecuados y factibles para su estudio(AU)

ABSTRACT Introduction: Mutations in the tyrosine kinase 3 gene (FLT3) are of special importance in acute myeloid leukemia because they serve as a guide to confirm the diagnosis, estimate the prognosis, and make therapeutic decisions in the patient. Internal tandem duplication (FLT3-ITD) is the most important alteration of this gene. Objective: To present the most relevant aspects regarding the FLT3-ITD biomarker in the context of acute myeloid leukemia. Methods: a search was carried out for updated scientific articles, in English and Spanish, in PubMed, EMBASE, Google Scholar and SciELO. Articles published in the last five years were selected. The most relevant aspects of the biomarker in the context of acute myeloid leukemia, its biological basis, the impact of the size of the fragments and the allelic load in the estimation of the prognosis of the patients, the new therapeutic strategies and the challenges in regarding the laboratory methods for its diagnosis. Information analysis and synthesis: Beyond the biomarker positivity or not, the size of the ITD, as well as the allelic ratio determined by the mutated allele / wild-type allele, could have a great impact on the prognosis of patients. However, differences persist in the criteria for establishing risk prediction algorithms, the cut-off point to be used as a reference, and the specific laboratory protocol for a more detailed study of the biomarker. Conclusions: The scientific community needs to continue working to clarify the practical utility of these parameters, validating them in broad and epidemiologically diverse series. The exact cut-off point should be determined as a reference to compare the relationship and standardize the most suitable and feasible laboratory methods for its study(AU)

Estudo de associação entre polimorfismos genéticos e níveis plasmáticos do VEGF, FLT1, KDR e o risco para depressão / Study of association between genetic polymorphisms and plasma levels of VEGF, FLT1, KDR and the risk for depression

Introdução: a depressão é um transtorno mental comum, grave e incapacitante que afeta mais de 350 milhões de pessoas em todo o mundo. A depressão é caracterizada principalmente por sintomas como tristeza, perda de interesse, diminuição da energia, perda de confiança e autoestima, culpa inadequada, distúrbios do sono e do apetite, pensamentos de morte e suicídio. Além disso, essa patologia também tem um forte impacto na qualidade de vida dos indivíduos afetados e de suas famílias. Sabe-se que fatores genéticos interagem com as condições socioambientais de modo a influenciar a predisposição das pessoas ao adoecimento. Estudos identificaram polimorfismos de nucleotídeos simples (SNPs) que podem ser marcadores genéticos apropriados para prever inflamação sistêmica, por exemplo, e a atual tese teve como foco o efeito de SNPs na via do fator de crescimento endotelial vascular (VEGF). Esta proteína é uma potente molécula angiogênica e está envolvida na neurogênese do hipocampo, uma das principais estruturas límbicas afetadas em pessoas com depressão. O VEGF está implicado em uma das principais teorias que tentam explicar a fisiopatologia deste transtorno mental grave, a teoria neurotrófica, a qual diz que a diminuição ou desregulação da sinalização de neurotrofinas pode contribuir para a manifestação do transtorno depressivo (TD). Objetivo: avaliar se polimorfismos do VEGF e seus receptores, KDR e FLT1, estão associados à depressão e à gravidade dos sintomas, à ideação e tentativas de suicídio, independentemente tanto de um tratamento otimizado quanto da presença de estresse precoce (do inglês, early-life stress, ELS), também verificar se há efeito destes polimorfismos nas concentrações plasmáticas de proteínas expressas pelos seus respectivos genes e observar se existe correlação entre VEGF e seus inibidores, VEGF e s100ß. Metodologia: participaram do presente estudo 160 pacientes com depressão e 114 controles saudáveis. Foram aplicados durante entrevista questionários que avaliaram o perfil clínico dos pacientes como o MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI e foi registrado o número de tentativas de suicídio. Os controles passaram por uma entrevista para serem avaliados quanto aos critérios de inclusão e exclusão do grupo. A genotipagem dos participantes foi realizada através da técnica de Real Time PCR e as mensurações de proteínas por meio do ensaio ensaio imunoenzimático (ELISA). Resultados: indivíduos com depressão, homozigotos AA do polimorfismo rs699947, apresentaram maiores concentrações plasmáticas de VEGF (P-valor= 0.006) e se associaram a um maior número de tentativas de suicídio na análise direta (P-valor= 0.041) e na análise corrigida foi observada uma tendência para a confirmação deste resultado (P-valor= 0.076). O genótipo homozigoto GG do polimorfismo rs7993418 do FLT1 se associou à severidade de sintomas (P-valor= 0.040), bem como uma tendência de associação com um aumento nas tentativas de suicídio e uma maior pontuação na escala que avaliou ideação suicida. Entre os pacientes quanto maior foram as concentrações plasmáticas de VEGF, maior foram as de KDR, FLT1 e s100ß. Conclusão: os resultados sugerem que os polimorfismos da via VEGF estão associados ao número de tentativas de suicídio e severidade dos sintomas depressivos

Introduction: Depression is a common, serious, and disabling mental disorder that affects more than 350 million people worldwide. Depression is mainly characterized by symptoms such as sadness, loss of interest, decreased energy, loss of confidence and self-esteem, inadequate guilt, sleep and appetite disturbances, thoughts of death and suicide. Furthermore, this pathology also has a strong impact on the quality of life of those affected and their families. It is known that genetic factors interact with social and environmental conditions to influence people's predisposition to illness. Studies have identified single nucleotide polymorphisms (SNPs) that may be appropriate genetic markers to predict systemic inflammation, for example, and the current thesis focused on the effect of SNPs on the vascular endothelial growth factor (VEGF) pathway. This protein is a potent angiogenic molecule and is involved in hippocampal neurogenesis, one of the main limbic structures affected in people with depression. VEGF is implicated in one of the main theories that try to explain the pathophysiology of this severe mental disorder, the neurotrophic theory, which says that the decrease or dysregulation of neurotrophin signaling can contribute to the manifestation of depressive disorder (DT). Objective: to assess whether polymorphisms of VEGF and its receptors, KDR and FLT1, are associated with depression and severity of symptoms, suicide ideation and attempts, regardless of both optimal treatment and the presence of early-life stress (ELS) in these associations. also check whether there is an effect of these polymorphisms on the plasma concentrations of proteins expressed by their respective genes and observe whether there is a correlation between VEGF and its inhibitors, VEGF and s100ß. Methodology: 160 patients with depression and 114 healthy controls participated in this study. Questionnaires that assessed the clinical profile of patients, such as the MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI, were applied during interviews, and the number of suicide attempts was recorded. The controls underwent an interview to be evaluated regarding the inclusion and exclusion criteria. The genotyping of the participants was performed using the Real Time PCR technique and protein measurements were performed using the enzyme-linked immunosorbent assay (ELISA). Results: individuals with depression, homozygous AA of the rs699947 polymorphism, had higher plasma concentrations of VEGF (P-value = 0.006) and a greater number of suicide attempts in the direct analysis (P-value = 0.041) and in the corrected analysis a trend towards confirmation of this result was observed (P-value = 0.076). The GG genotype of the FLT1 polymorphism rs7993418 was associated with symptom severity (P-value = 0.040), as well as with a trend for association with increase in suicide attempts and a higher score on the scale that evaluated suicidal ideation. The bigger the plasma concentrations of VEGF, the higher were those of KDR, FLT1 and s100ß. Conclusion: the results indicate that VEGF pathway polymorphisms are associated with the number of suicides and severity of depressive symptoms

Progress of targeted therapy for acute myeloid leukemia / 白血病·淋巴瘤

The treatment of acute myeloid leukemia (AML) has made a great progress in recent years owing to the emergence of targeted drugs. While the problems such as drug resistance, the increasing incidence of adverse reactions of the combined therapies, the lack of the effective therapy targets became growing concerns with the further development of studies, which has posed a big challenge to the therapy strategies of AML. This article introduces the progress of the latest clinical trial outcomes in targeted therapy for AML at the 63rd American Society of Hematology (ASH) annual meeting which reported some hot issues like options for the treatment timing of targeted therapy, the selection for the drugs and the combined drugs.

Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML / 中国实验血液学杂志

OBJECTIVE@#Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.@*METHODS@#The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.@*RESULTS@#In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.@*CONCLUSION@#The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Association of Next Generation Sequencing Based Genotypic Profiling with MICM Characteristics in NPM1 Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explain the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) by analyzing the association between next-generation sequencing (NGS) profiles and MICM characteristics in patients with this AML subtype.@*METHODS@#Data of 238 NPM1mut patients with available NGS information on 112 genes related to blood disease was collected, and χ2 test and nonparametric test were used to analyze the distribution association between NGS-detecting mutations and conventional MICM parameters.@*RESULTS@#In entire NPM1mut cohort, totaling 240 NPM1 mutation events were identified, of whom 10 (10/240, 4.2%) were missense mutations, which did not involve any W288 or W290 locus and were found exclusively in NPM1mut/FLT3-ITD- group. All but one of these missense mutations (9/10, 90%) were accompanied by AML subtype-defining recurrent cytogenetic or molecular abnormalities, of which 7 cases were in the low risk and 2 in the high risk. NPM1mut occurred solely as an insertion/deletion (indel) type in the NPM1mut/FLT3-ITD+ group. The incidence of favorable plus unfavorable karyotypes in NPM1mut/FLT3-ITD- group was higher than in NPM1mut/FLT3-ITD+ group (6.4% vs. 0, P=0.031). The positive rates of CD34 and CD7 in NPM1mut/FLT3-ITD+ group were significantly higher than in NPM1mut/FLT3-ITD- group (CD34: 47.9% vs. 20.6%, P<0.001; CD7: 61.5% vs. 29.9%, P<0.001). Logistic analysis showed that FLT3-ITD independently predicted for CD34+ and CD7+ [odds ratio (OR)=5.29, 95%CI: 2.64-10.60, P<0.001; OR=3.47, 95%CI: 1.79-6.73, P<0.001; respectively]. Ras-pathway mutations independently predicted for HLA-DR+ (OR=4.05, 95%CI: 1.70-9.63, P=0.002), and KRAS mutation for MPO- (OR=0.18, 95%CI: 0.05-0.62, P=0.007). TET2/IDH1 mutations independently predicted for CD34- and CD7- (OR=0.26, 95%CI: 0.11-0.62, P=0.002; OR=0.30, 95%CI: 0.14-0.62, P=0.001; respectively), and MPO+ (OR=3.52, 95%CI: 1.48-8.38, P=0.004). DNMT3A-R882 independently predicted for CD7+ and HLA-DR+ (OR=3.59, 95%CI: 1.80-7.16, P<0.001; OR=13.41, 95%CI: 4.56-39.45, P<0.001; respectively), and DNMT3A mutation for MPO-(OR=0.35, 95%CI: 1.48-8.38, P=0.004).@*CONCLUSION@#Co-existing FLT3-ITD in NPM1mut AML independently predicts for CD34+ and CD7+, co-existing Ras-pathway mutation for HLA-DR+ and MPO-, co-existing TET2/IDH1 mutation for CD34-, CD7-, and MPO+, and co-existing DNMT3A mutation for HLA-DR+, CD7+, and MPO-, thereby providing a new mechanism explanation for the immunophenotypic heterogeneity of these AML patients.

Effects of Paclitaxel and Quizartinib Alone and in Combination on AML Cell Line MV4-11 and Its STAT5 Signal Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of paclitaxel, quizartinib and their combination on proliferation, apoptosis and FLT3/STAT5 pathway of human leukemia cell line MV4-11 (FLT3-ITD+).@*METHODS@#MV4-11 cells were treated with paclitaxel and quizartinib at different concentrations for 24 h, 48 h and 72 h, respectively, and then the two drugs were combined at 48 h to compare the inhibition of proliferation, the apoptosis rate was detected by flow cytometry, the expression of FLT3 and STAT5 mRNA was determined by fluorescence quantitative PCR, and the protein expression of FLT3, p-FLT3, STAT5 and p-STAT5 was determined by Western blot.@*RESULTS@#Different combination groups of paclitaxel and quizartinib had synergistic inhibitory effect. The cell survival rate in the combination group was significantly lower than that in the single drug group (P<0.05). The cell apoptosis rate in the combination group was significantly higher than that in the single drug group (P<0.001). The expression of FLT3 mRNA in combination group was significantly higher than that in two single drugs (P<0.01). The expression of STAT5 mRNA in combination group was significantly higher than that in quizartinib group (P<0.001); increased compared with paclitaxel group, but there was no statistical significance. The expression level of p-FLT3、p-STAT5 protein in the combination group was significantly lower than that in the single drug group (P<0.05, P<0.05).@*CONCLUSION@#Paclitaxel combined with quizartinib can synergistically inhibit the proliferation of MV4-11 cell line and promote the apoptosis of MV4-11 cell line by inhibiting the activity of FLT3/STAT5 pathway.

Clinical Correlation of Lactate Dehydrogenase Activity with JAK2V617F and FLT3 Mutations in Myeloproliferative Disorders and Acute Myeloid Leukemia: Indian Population

Background: Lactate dehydrogenase (LDH) play main role in the cell damage.Previous study has shown elevated level in acute leukemia and myeloproliferative disorders. Molecular markers specifically for JAK2V617F mutation in myeloproliferative disorders (MPDs) and FLT3 mutations in acute myeloid leukemia patients (AML) are well known for clinical practice. But, still only few reports have shown the correlation of LDH activity with molecular markers in Hematological Malignancy. Therefore, aim of study is to correlate LDH activity with established specific molecular markers in MPDs and AML. Methods:10 healthy individuals, 50 patients with MPDs and 50 patients with AML were included for the study. Serum LDH was performed by autoanalyser. The JAK2V617F mutations is analyse from blood by Real time PCR and FLT3 ITD/D835 mutations are analysed from blood by PCR-RFLP respectively. Statistical analysis was done by SPSS statistical software. Results: LDH Activity were higher in MPDs and AML as compared with healthy individual. Mean LDH activity was higher in FLT3 ITD/D835 positive mutation and JAK2V617F positive mutation in AML and MPDs respectively was observed. Conclusion: High LDH activity with Positive frequency of FLT3 ITD/D835 mutation in Acute Myeloid Leukemia and Positive frequency of JAK2V617F mutation in Myeloproliferative Disorders were observed.

The Role of FLT3-ITD and CCAAT-Enhancer-Binding Protein α Mutations on Prognosis of Acute Lymphoblastic Leukaemia in Turkish Patients

ABSTRACT Background: Acute lymphoblastic leukaemia (ALL) is the most common malignancy in childhood. Although some prognostic factors have been defined to date, the estimation of prognosis is currently not perfect. Previous studies had shown an association of FLT3 with poor prognosis and CCAAT-enhancer-binding protein α (CEBPA) mutation with the development of acute myeloid leukaemia (AML). Here, we aimed to evaluate the prognostic value of FLT3-ITD and CEBPA mutations in ALL. Methods: Sixty-one patients with ALL were included in the study. The patients were divided into three risk groups according to BFM risk classification. All of the patients were examined for FLT3-ITD mutations and 45 of them for CEBPA mutations. Mutation positive and negative patients were compared in terms of their risk groups, translocations and cell lineage. The clinical courses of the patients were appraised. Results: FLT3-ITD mutation was detected in 3 of the 61 patients, and CEBPA mutations were detected in 11 of the 45 patients. The incidence of established prognostic indicators including BFM risk classification, t(9; 22); BCR-ABL, t(1; 19); E2A-PBX1, t(12; 21); TEL-AML1, t(4; 11); MLL-AF4 were similar between FLT3-ITD and CEBPA positive and negative patients. A patient with an FLT3-ITD mutation was very susceptible to pancytopenia after maintenance treatment and two other patients with FLT3-ITD mutations were more prone to febrile neutropenia. Conclusion: Our results suggested that CEBPA or FLT3-ITD mutations might not be related to ALL prognosis in the sampled Turkish patients. However, FLT3-ITD mutation might have an influence on the response of bone marrow to chemotherapy.

Diagnostic value of Flt3L and Gas6 in post-chemotherapy infection in patients with non-Hodgkin′s lymphoma / 中华检验医学杂志

Objective:To investigate the diagnostic value of Flt3 ligand (Flt3L) and growth arrest specific protein 6 (Gas6) in post-chemotherapy infection of patients with non-Hodgkin′s lymphoma.Methods:A total of 94 NHL patients admitted to Wuhan No.1 Hospital for chemotherapy from July 2019 to October 2020 were collected, and divided into infection group ( n=40) and non-infection group ( n=54). Fifty healthy subjects at the same period were selected as the control group to analyze the expression levels of Flt3L, Gas6 and general inflammatory indicators among the three groups. The relationship between Flt3L, Gas6 and general inflammatory indicators were analyzed. The predictive value of Flt3L and Gas6 for post-chemotherapy infection in NHL patients was analyzed by ROC curve. Kaplan-Meier curve was used to analyze the infection status of NHL chemotherapy patients with serum Flt3L and Gas6 levels. Results:The levels of serum Flt3L [807.80(215.10-1 232.00) pg/ml] and Gas6 [20.04(13.14-27.52) ng/ml] in the infection group were higher than those in the non-infection group and the control group, and the differences were statistically significant (all P<0.05);The levels of serum Flt3L[887.3(321.60-1 367.00) pg/ml] and Gas6[25.24(17.61-42.86) ng/ml] in the severely infection group were higher than those in the mildly infection group, and the differences were statistically significant (all P<0.05). serum Flt3L was negatively correlated with WBC and positively correlated with IL-6;serum Gas6 was negatively correlated with WBC.ROC curve analysis showed that the AUC and 95% CI of Flt3L and Gas6 were 0.814 (0.718-0.909) and 0.644 (0.523-0.765), respectively. The combined application of serum Flt3L with hsCRP, PCT and IL-6 significantly improved the diagnostic efficiency, and the area under the curve was 0.956, 0.923 and 0.865, respectively. Kaplan-Meier curve analysis showed that when serum Flt3L≥649.80 pg/ml and serum Gas6≥21.50 ng/ml in NHL chemotherapy patients, the infection rate was significantly increased ( P all<0.05). Conclusion:Serum Flt3L and Gas6 levels in post chemotherapy-infection of NHL patients were significantly increased and correlated with the severity of infection. Early detection of serum Flt3L levels is helpful to predict the diagnosis of infection in NHL patients after chemotherapy.

Identificación de alteraciones moleculares en pacientes Venezolanos con diagnóstico de leucemia linfoblástica aguda / Identification of molecular alterations in Venezuelan patients with acute lymphoblastic leukemia diagnosis

La Leucemia Linfoblástica Aguda (LLA) es la neoplasia más frecuente en edad pediátrica. En los últimos años, entre el 15 y 20% de los pacientes fracasan en el tratamiento. Conocimientos en citogenética y biología molecular repercuten de manera importante en la determinación del pronóstico y del esquema de tratamiento adecuado. En Venezuela existe un conocimiento limitado en cuanto a la genética molecular de esta alteración onco-hematológica. El objetivo del trabajo fue evaluar las alteraciones genéticas más frecuentes en pacientes venezolanos con diagnóstico clínico de leucemia linfoblástica aguda. Se realizó un estudio transversal, descriptivo y prospectivo de 2006 a 2014, en el que se evaluaron las translocaciones ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, así como las mutaciones en los genes PAX5 y FLT3 mediante el uso de diferentes tipos de PCR. Ciento treinta pacientes con diagnóstico clínico de leucemia linfocítica aguda fueron incluidos en el estudio. Se identificaron alteraciones moleculares en 56 pacientes (43,1%), en los que observamos la presencia de una o varias alteraciones en conjunción en un mismo paciente. Las alteraciones identificadas fueron t(12;21) (11,5%), t(4;11) (8,5%), t(1;19) (10%), t(9;22) (20,8%), ITD-FLT3 (14,8%), mutación P80S (4,2%) y S77del (4,2%) en el gen PAX5. La prevalencia de BCR/ ABL, es una de las más altas que ha sido descrita hasta ahora en casos de LLA donde la mayor parte de la población está conformada por pacientes pediátricos. Estos resultados representan el primer estudio molecular de la LLA en Venezuela, sentando las bases para el diagnóstico y seguimiento de la enfermedad en su población.

Acute Lymphoblastic Leukemia (ALL) is the most common neoplasm in pediatric age. In recent years, between 15 and 20% of patients failed in their treatments. Knowledge on cytogenetics and molecular biology has an important impact on the determination of the prognosis and the appropriate treatment scheme. In Venezuela there is limited knowledge regarding the molecular genetics of this onco-hematological alteration. The aim of this work was to evaluate the most frequent genetic alterations in Venezuelan patients with a clinical diagnosis of acute lymphoblastic leukemia. A cross-sectional, descriptive and prospective study was carried out from 2006 to 2014, in which the translocations ETV6/RUNX1, MLL/AF4, TCF3/PBX1, BCR/ABL1, as well as mutations in the PAX5 and FLT3 genes were evaluated through the use of different types of PCR. One hundred and thirty patients with a clinical diagnosis of acute lymphocytic leukemia were included in the study. Molecular alterations were identified in 56 patients (43.1%), in which we observed the presence of one or several alterations in conjunction in the same patient. The alterations identified were t(12; 21) (11.5%), t(4; 11) (8.5%), t(1; 19) (10%), t(9; 22) (20.8%), ITD-FLT3 (14.8%), P80S mutation (4.2%) and S77del (4.2%) in the PAX5 gene. The prevalence of BCR/ABL is one of the highest described so far in cases of ALL where most of the population is made up of pediatric patients. These results represent the first molecular study of ALL in Venezuela, laying the foundations for the diagnosis and monitoring of the disease in its population.

Favorable long-term survival using consolidation chemotherapy without allogeneic hematopoietic cell transplantation for acute myeloid leukemia with wild-type NPM1 without FLT3-ITD

BACKGROUND: The role of allogeneic hematopoietic cell transplantation (allo-HCT) compared with consolidation chemotherapy alone in intermediate-risk acute myeloid leukemia (AML) patients with wild-type nucleophosmin/negative or a low level of Fms related tyrosine kinase 3 internal tandem duplication (NPM1(wt)/FLT3-ITD(neg/low)) has not yet been elucidated. METHODS: In this study, we retrospectively investigated 88 patients newly diagnosed with AML who received intensive induction chemotherapy at Kyungpook National University Hospital from March 2015 to July 2017. The selection criteria included the presence of results on genetic abnormalities including NPM1 and FLT3-ITD. RESULTS: According to the European LeukemiaNet (ELN) risk classification, 25 patients (28%) were categorized as favorable, 44 (50%) as intermediate, and 19 (22%) as adverse risk. Among the intermediate-risk patients, 40 were identified as NPM1 wt/FLT3-ITDneg/low. Among the patients with NPM1(wt)/FLT3-ITD(neg/low), complete remission (CR) was achieved in 26 patients out of 40 (65%). One-year overall survival (OS) rate was 100% in the favorable-risk group and 87.9% in the NPM1(wt)/FLT3-ITD(neg/low) group (P=0.233). Among the intermediate-risk NPM1(wt)/FLT3-ITD(neg/low) patients, there was no survival benefit with allo-HCT (N=19) compared to consolidation chemotherapy (N=21; P=0.372). In the multivariate analysis, the ELN risk group [hazard ratio (HR), 6.36; P=0.019] and the achievement of CR (HR, 2.95; P=0.017) were both identified as factors affecting OS of patients with newly diagnosed AML. CONCLUSION: Among the AML patients, intermediate-risk NPM1(wt)/FLT3-ITD(neg/low) patients and favorable-risk patients showed similar OS rates. Our results suggested that allo-HCT might have limited clinical benefit for the intermediate-risk NPM1(wt)/FLT3-ITD(neg/low) patients. Well controlled studies are needed to confirm the current results.

Influences on induction and maturation of mouse bone marrow-derived dendritic cells by varying combinations of cytokines / 中华微生物学和免疫学杂志

Objective To investigate the influences of culture conditions on the in vitro induction and maturation of dendritic cells by using different combinations of cytokines. -ethods Mouse bone mar-row cells were isolated and cultured in media containing varying combinations of cytokines, including granu-locyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and fms-like tyrosine kinase 3 ligand (Flt3L). After cultured at 37℃ for seven days, the attached bone marrow cells were collected and stained by fluorescence-labeled monoclonal antibodies (McAb) against CD11c, MHCⅡ and CD86 for flow cytometry analysis. In the parallel group, LPS was added on day 5 to a final concentration of 1μg/ml for DC maturation analysis by flow cytometry. Results In group Flt3L (20 ng/ml)/GM-CSF (20 ng/ml)/IL-4 (10 ng/ml), 90％ of bone marrow cells were CD11c-positive. Flt3L (100 ng/ml) could induce 88％ of bone marrow cells to express CD11c. Bone marrow cells positive for MHCⅡ accounted for 35. 4％ and 36. 1％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF, where both Flt3L and GM-CSF were used at a concentration of 20 ng/ml. After LPS stimulation, the positive rates of MHCⅡ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF were 58. 1％ and 59. 6％, which increased by 22. 7％ and 23. 5％, re-spectively. The percentages of CD86-positive bone marrow cells were 7. 1％ and 5. 5％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF. Bone marrow cells positive for CD86 grew by 7. 1％ and 6. 2％ in group Flt3L (20 ng/ml) and group GM-CSF/IL-4 after LPS stimulation. Conclusions Flt3L and GM-CSF probably dominated the differentiation and maturation of bone marrow-derived dendritic cells with a synergis-tic effect. Combined usage of Flt3L and GM-CSF at the concentration of 20 ng/ml would be an optimal proto-col for DC research.

Analysis of induction efficacy and prognostic factors in FLT3-ITD positive acute myeloid leukemia in the real world / 中华血液学杂志

Objective: To investigate the efficacy and prognostic factors of induction therapy in FLT3-ITD(+) acute myeloid leukemia (AML) in the real world data. Methods: From January 2013 to December 2016, 114 de novo patients with FLT3-ITD(+)AML were enrolled in this study. Out of 114 cases, 75 were male, and 39 were female. The median age was 42 years old (ranged from 14 to 72 years old) . The chemotherapy regimens were used for induction therapy and all cases were followed up. The treatment response was evaluated by MICM and the comparison of the ratio were analyzed by chi-square test and the survival was estimated by Kaplan-Meier analysis and Cox proportional hazards model was used to identify independent prognostic factors. Results: There were 52 FLT3-ITD(+)AML patients with favorable prognosis genes (46 cases with NPM1, 5 cases with RUNX1-RUNX1T1, 1 case with CEBPA double mutation) and 62 patients with other types of FLT3-ITD(+)AML at diagnosis. All patients completed at least one cycle of induction therapy and the clinical curative effect was evaluated, complete remission (CR) rate was 50.0% (57/114) in one cycle and total CR rate was 72.5% (74/104) in two cycles. The CR rate of the FLT3-ITD(+) AML patients with favorable prognosis genes was 67.3% (35/52) in one cycle and 83.3% (40/48) in two cycles; for the other types FLT3-ITD(+)AML patients, the CR rate was 35.5% (22/62) in one cycle and 64.8% (35/54) in two cycles. There was a significant difference in CR rate between the FLT3-ITD(+)AML patients with and without favorable prognosis genes (P<0.05) . This indicates that the FLT3-ITD(+)AML patients with favorable prognosis gene had relatively good therapeutic effect. Among other types of FLT3-ITD(+)AML patients who did not achieve remission from one cycle of chemotherapy, 9 patients were given sorafenib plus chemotherapy and 6 cases (66.7%) achieved CR; 23 patients were given conventional chemotherapy and 7 cases (30.4%) achieved CR. There was a significant difference between sorafenib plus chemotherapy and conventional chemotherapy groups (χ(2)=4.47, P<0.05) and this indicates that sorafenib plus chemotherapy can significantly improve the CR rate of FLT3-ITD(+)AML patients. Comparing overall survival (OS) and disease free survival (DFS) , there was no significant difference between sorafenib plus chemotherapy and conventional chemotherapy groups (P values were 0.641 and 0.517, respectively) . Conclusion: The overall prognosis of FLT3-ITD(+)AML patients is poor, and the stratification therapeutic efficacy of FLT3-ITD(+)AML without favorable prognosis gene can be improved by sorafenib combined with chemotherapy.