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.

Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1-mutated acute myeloid leukemia

Although NPM1 mutations are frequently found in acute myeloid leukemia patients, therapeutic strategies are scarce and unsuitable for those who cannot tolerate intensive chemotherapy. Here we demonstrated that heliangin, a natural sesquiterpene lactone, exerts favorable therapeutic responses in NPM1 mutant acute myeloid leukemia cells, with no apparent toxicity to normal hematogenous cells, by inhibiting their proliferation, inducing apoptosis, causing cell cycle arrest, and promoting differentiation. In-depth studies on its mode of action using quantitative thiol reactivity platform screening and subsequent molecular biology validation showed that the ribosomal protein S2 (RPS2) is the main target of heliangin in treating NPM1 mutant AML. Upon covalent binding to the C222 site of RPS2, the electrophilic moieties of heliangin disrupt pre-rRNA metabolic processes, leading to nucleolar stress, which in turn regulates the ribosomal proteins-MDM2-p53 pathway and stabilizes p53. Clinical data shows that the pre-rRNA metabolic pathway is dysregulated in acute myeloid leukemia patients with the NPM1 mutation, leading to a poor prognosis. We found that RPS2 plays a critical role in regulating this pathway and may be a novel treatment target. Our findings suggest a novel treatment strategy and lead compound for acute myeloid leukemia patients, especially those with NPM1 mutations.

Effect of negative regulation of nucleophosmin 1 by long non-coding RNA growth arrest specific transcript 5 on cisplatin resistance of gastric cancer cells / 中国生物制品学杂志

@#Objective To investigate the effects of long non-coding RNA(LncRNA) growth arrest specific transcript 5(GAS5) negatively regulating nucleophosmin 1(NPM1) on cisplatin(DDP) resistance of gastric cancer cells.Methods The normal human gastric mucosa cell line GES-1 and human gastric cancer cell lines BG3-823,MGC-803 and AGS were selected as the research objects,of which the level of LncRNA GAS5 in each cell was measured by qRT-PCR.The drug resistance of AGS cells to DDP(AGS/DDP) was induced,and the experiment was divided into control group,empty plasmid group(BC group),GAS5 nonsense interference group(pLJM-GAS5 NC group) and GAS5 overexpression group(pLJM-GAS5 group).MTT method was used to determine the effect of DDP on the proliferation of AGS and AGS/DDP cells;and the levels of NPM1,multidrug resistance 1(MDR1),excision repair cross complementation group 1(ERCC1),multidrug resistance-associated protein 1(MRP1) and N-cadherin in AGS and AGS/DDP cells were measured by Western blot.Results Compared with the normal gastric mucosa GES-1 cells,the level of LncRNA GAS5 in BG3-823 and AGS cells decreased significantly,and among them,the level of LncRNA GAS5 in AGS cells was the lowest,so AGS cells were used for the follow-up experiments.Compared with the control group,the level of LncRNA GAS5 in AGS cells of BC group and pLJM-GAS5 NC group decreased significantly,while the levels of NPM1,MDRl,ERCC1,MRP1 and N-cadherin increased significantly;compared with BC group and pLJM-GAS5 NC group,the level of LncRNA GAS5 in AGS/DDP cells of pLJM-GAS5 group increased significantly,while the levels of NPM1,MDR1,ERCC1,MRP1 and N-cadherin decreased significantly;after treatment with DDP of the same concentration(except 0 μmol/L),compared with the control group,the inhibition rate of AGS/DDP cell proliferation in BC group and pLJM-GAS5 NC group decreased significantly;compared with BC group and pLJM-GAS5 NC group,the inhibition rate of AGS/DDP cell proliferation in pLJM-GAS5group was significantly higher.The semi inhibitory concentration(IC_(50)) of DDP on AGS/DDP cells in pLJM-GAS5 group for 48 h was(65.38±5.04) μmol/L,which was significantly lower than(120.74±4.17) μmol/L and(120.24±4.29) μmol/L in BC group and pLJM-GAS5 NC group.Conclusion Up-regulating the level of LncRNA GAS5 in AGS/DDP cells can reverse the drug resistance of AGS/DDP cells,which may be related to the down-regulation of NPM1expression

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.

Effect of clonal hematopoiesis in remission on hematopoiesis recovery in patients with NPM1 mutated acute myeloid leukemia after chemotherapy / 中华血液学杂志

Objective: To investigate the effect of clonal hematopoiesis (CH) in remission on hematopoiesis recovery in patients with NPM1 mutated acute myeloid leukemia (AML) after chemotherapy. Methods: Retrospective analysis was performed on 86 patients with NPM1(mut) AML newly diagnosed and treated in the First Affiliated Hospital of Soochow University between July 2016 and June 2019. Their clinical data and NGS test results at diagnosis were analyzed. Moreover, bone marrow samples in remission were tested using Sanger sequencing. The log-rank test was used to analyze the difference in hematopoietic recovery, and Cox proportional hazard models were used to analyze the prognostic factors affecting hematopoietic recovery. Results: The median age of the 86 NPM1(mut) AML patients was 50 years (15-69 years). There were 39 males and 47 females. Forty-one patients were induced with intensity chemotherapy ("7 + 3"), whereas 45 patients were treated with low-dose cytarabine-based induction chemotherapy. At diagnosis, The most common mutations in the patients were FLT3, DNMT3A, TET2, and IDH1/IDH2 mutations. CH-associated mutations persisted in 21 patients during remission, and the mutations were DNMT3A, TET2, ASXL1, and IDH1/IDH2. The recovery time of neutrophils in patients with CH-associated mutations in remission was consistent with that in patients without CH in remission (P=0.282) but the recovery time of platelets in patients with CH in remission was significantly longer[26 (95% CI 21-32) days vs 25 (95% CI 23-26) days, P=0.032]. Furthermore, univariate analysis indicated that age, induced chemotherapy program, and CH in remission were risk factors for platelet recovery, whereas multivariate analysis indicated that induced chemotherapy program and CH in remission were independent risk factors for platelet recovery (HR=0.454, P=0.001 and HR=0.520, P=0.027, respectively) . Conclusion: CH in remission delays the hematopoietic recovery of patients with NPM1(mut) AML after chemotherapy.

Clinical Significance of Low Expression of LncRNA CASC15 in Acute Myeloid Leukemia with NPM1 Mutations / 中国实验血液学杂志

AbstractObjective: To identify the expression and methylation patterns of lncRNA CASC15 in bone marrow (BM) samples of acute myeloid leukemia (AML) patients, and further explore its clinical significance.@*METHODS@#Eighty-two de novo AML patients and 18 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the expression and methylation data of CASC15. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of CASC15 expression to identify AML. The patients were divided into CASC15high group and CASC15low group by X-tile method, and the prognostic value of CASC15 was identified by Kaplan-Meier method and univariate and multivariate Cox regression analysis.@*RESULTS@#The expression level of CASC15 was significantly decreased in BM cells of AML patients compared with healthy donors (P<0.001). ROC curve analysis suggested that CASC15 expression might be a potential biomarker to discriminate AML from controls. The expression of CASC15 was high at the early stage of hematopoiesis, and reached a peak at the stage of multipotent progenitors differentiation, then decreased rapidly, and was at a range of low level fluctuations in the subsequent process. Among FAB subtypes, CASC15 expression in M0 was significantly higher than that in M1-M7. Clinically, CASC15low patients were more likely to have NPM1 mutations than CASC15high patients (P=0.048), while CASC15high patients had a significantly higher frequency of IDH1 and RUNX1 mutations (P=0.021 and 0.014, respectively). Moreover, CASC15low group had a shorter overall survival (OS) in patients with NPM1 mutations. Furthermore, multivariate analysis confirmed that CASC15 expression was a significant independent risk factor for OS in NPM1 mutated AML patients. In addition, CASC15 methylation level in BM samples of AML patients was significantly decreased compared with healthy donors. Patients with CASC15 high methylation had poor OS and disease-free survival.@*CONCLUSION@#The expression of CASC15 is decreased in AML, and low CASC15 expression may predict adverse prognosis in AML patients with NPM1 mutations. Moreover, CASC15 methylation level in AML is significantly decreased, and high CASC15 methylation may predict poor prognosis in AML.

Detection of NPM1 Mutation in Acute Myeloid Leukemia by Droplet Digital PCR and Its Clinical Application Value / 中国实验血液学杂志

OBJECTIVE@#To establish the droplet digital PCR (ddPCR) assay for the detection of NPM1 type A mutation in patients with acute myeloid leukemia (AML), and to evaluate its specificity, sensitivity and its value in clinical application.@*METHODS@#NPM1 mutant and wildtype plasmids were used to verify the performance of ddPCR. Both ddPCR and Sanger sequencing were used to detect the bone marrow samples of 87 AML patients, which were confirmed by next generation sequencing (NGS). Moreover, NPM1 mutation burden was dynamically monitored in five patients by ddPCR.@*RESULTS@#The limit of blank (LOB) of ddPCR established for NPM1 mutation detection was 1.1 copies/μl, and the limit of detection (LOD) was 2.43 copies/μl, which had good linearity. Among the 87 newly diagnosed AML patients, ddPCR identified seventeen cases positive for NPM1 mutation (19.5%)， which was consistent with Sanger sequencing. NGS confirmed 12 positive cases, including 8 of type A mutations, 2 of type D mutations, and 2 of rare type mutations. The results of dynamic monitoring of NPM1 mutation burden in 5 patients showed that the NPM1 mutation burden decreased obviously even close to 0, when patients achieve complete remission after chemotherapy. However, the mutation burden was increased again at the time of relapse.@*CONCLUSION@#In this study, we established a ddPCR method for detection of NPM1 mutation with good sensitivity and repeatability, which can be used for screening NPM1 mutation in newly diagnosed AML patients and for minimal residual disease monitoring after remission in positive AML patients to guide treatment.

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.

Caso atípico de leucemia mieloide aguda con coexistencia de NPM1-A e inversión del cromosoma 16 / A rare case of acute myeloid leukemia with coexistence of NPM1-A mutation and chromosome 16 inversion

Introducción: La leucemia mieloide aguda (LMA) es un grupo heterogéneo de desórdenes clonales con una gran variabilidad en términos de patogénesis, características morfológicas, genéticas e inmunofenotípicas. Las mutaciones en el gen NPM1 representan una de las más comunes en las LMA y está asociada con una respuesta clínica favorable. Por citogenética, la inversión del cromosoma 16 define el subgrupo de las LMA de factor de unión al grupo con un pronóstico favorable. Objetivo: Describir un caso con diagnóstico de LMA en los cuales el estudio molecular del gen NPM1 y de la inv(16) fueron positivos. Caso clínico: A nivel molecular, la hibridación in situ fluorescente fue positivo a la inv(16) y por biología molecular fue positivo tanto a la inv(16) como al gen NPM1-A, elementos de baja frecuencia de aparición. Se le administró a la paciente un esquema de poliquimioterapia no intensiva para mejorarla clínicamente. Después de una mejoría clínica inicial, la paciente comenzó con complicaciones y falleció. Conclusiones: La coexistencia de estas dos mutaciones es muy poco frecuente en pacientes con LMA, y a pesar de ser de buen pronóstico la paciente falleció a los pocos días de tratamiento(AU)

Introduction: Acute myeloid leukemia (AML) is a heterogeneous group of clonal disorders with great variability in terms of pathogenesis, morphological, genetic and immunophenotypic characteristics. NPM1 mutations represent one of the most common in AML and are associated with favorable clinical response. By cytogenetics, chromosome 16 inversion defines, with a favorable prognosis, the core‐binding factor for the subgroup of AMLs Objective: To describe a AML case in which the molecular study of the NPM1 gene and the chromosome 16 inversion were positive. Clinical case: At the molecular level, fluorescent in situ hybridization was positive for chromosome 16 inversion and, by molecular biology, it was positive for both chromosome 16 inversion and for the NPM1-A gene, elements with a low frequency of appearance. The patient was administered a non-intensive combination as part of a chemotherapy regimen to improve her clinical status. After initial clinical improvement, the patient began with complications and died. Conclusions: The coexistence of these two mutations is very rare in patients with AML. Despite presenting a good prognosis, the patient died after a few days of treatment(AU)

Effect of NPM1 mutant A on TGF-β1-induced K562 cell proliferation and AKT phos-phorylation / 中国肿瘤临床

Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.

Value of molecular genetics combined with bone marrow imaging detection in the prognosis judgement of acute myeloid leukemia with normal karyotype / 白血病·淋巴瘤

Objective@#To investigate the value of NPM1 and FLT3 gene mutation combined with bone marrow imaging detection in the prognosis judgement of initial treatment cytogenetically normal acute myeloid leukemia (CN-AML).@*Methods@#The clinical data of 100 patients (non-M3 type) with primary and initial treatment CN-AML from January 2010 to January 2014 in the Peace Hospital Affiliated of Changzhi Medical College were retrospectively analyzed. All patients were enrolled in the bone marrow imaging examination on the end day of induction treatment or the first day after the end of induction treatment (T time point). Univariate and multivariate prognostic analyses were performed on AML patients according to FLT3 and NPM1 gene status,bone marrow juvenile cell ratio at T time point.@*Results@#A total of 100 patients included 36 cases with FLT3 gene mutation and 44 cases with NPM1 gene mutation. The complete remission (CR) rate of CN-AML patients was 13.9% (5/36) and 71.9% (46/64), respectively (P < 0.01), 2-year recurrence-free survival (RFS) rate was 5.6% and 59.8%, respectively (P < 0.01), 2-year overall survival (OS) rate was 15.6% and 66.2%, respectively in FLT3+ group and FLT3- group (P < 0.01). The median RFS and median OS time in FLT3+ group was 6.9 months and 9.4 months, respectively, and the median RFS and median OS time in FLT3- group had not yet reached. There were no significant differences of all the indexes between the two groups (all P < 0.01). And there were no significant differences in CR rate, 2-year RFS rate and 2-year OS rate between NPM1+ group and NPM1- group (all P > 0.05). The CR rate, 2-year RFS rate and 2-year OS rate in NPM1+ FLT3- group were better than those in NPM1- FLT3-, NPM1- FLT3+ and NPM1+ FLT3+ groups; NPM1- FLT3+ and NPM1+ FLT3+ groups had the worst prognosis, and there were no statistical differences in the CR rate, RFS and OS time between the two groups (all P > 0.05). The prognosis of the patients in bone marrow juvenile cell ratio < 0.05 group at T time point was better than that in ratio ≥0.05 group (all P < 0.05). CN-AML patients were classified into the good prognosis group (NPM1- FLT3-), the medium prognosis group (NPM1+ FLT3-), and the poor prognosis group (NPM1- FLT3+ and NPM1+ FLT3+) according to the FLT3 and NPM1 genes. The good prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group, the good prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥0.05 group, the medium prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group had no statistical differences in CR rate, 2-year RFS rate and 2-year OS rate (all P > 0.05). The medium prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥0.05 group, the poor prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group had the equivalent prognosis, and the average prognosis was moderate; the poor prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥ 0.05 group had the worst prognosis. According to Cox multivariate regression analysis, FLT3 gene mutation and the ratio of bone marrow juvenile cells at T time point were independent influencing factors for RFS and OS in CN-AML patients (all P < 0.05). NPM1 was an independent prognosis factor affecting RFS and OS of FLT3- patients (all P < 0.05).@*Conclusions@#After induction chemotherapy, the responsiveness and sensitivity of AML patients to chemotherapy regimen can be assessed early and objectively according to molecular genetics and the ratio of bone marrow juvenile cells at T time point, which has a certain value in the prognosis judgement.

Value of molecular genetics combined with bone marrow imaging detection in the prognosis judgement of acute myeloid leukemia with normal karyotype / 白血病·淋巴瘤

Objective To investigate the value of NPM1 and FLT3 gene mutation combined with bone marrow imaging detection in the prognosis judgement of initial treatment cytogenetically normal acute myeloid leukemia (CN-AML). Methods The clinical data of 100 patients (non-M3 type) with primary and initial treatment CN-AML from January 2010 to January 2014 in the Peace Hospital Affiliated of Changzhi Medical College were retrospectively analyzed. All patients were enrolled in the bone marrow imaging examination on the end day of induction treatment or the first day after the end of induction treatment (T time point). Univariate and multivariate prognostic analyses were performed on AML patients according to FLT3 and NPM1 gene status, bone marrow juvenile cell ratio at T time point. Results A total of 100 patients included 36 cases with FLT3 gene mutation and 44 cases with NPM1 gene mutation. The complete remission (CR) rate of CN-AML patients was 13.9% (5/36) and 71.9% (46/64), respectively (P< 0.01), 2-year recurrence-free survival (RFS) rate was 5.6% and 59.8%, respectively (P< 0.01), 2-year overall survival (OS) rate was 15.6% and 66.2%, respectively in FLT3+group and FLT3-group (P<0.01). The median RFS and median OS time in FLT3+ group was 6.9 months and 9.4 months, respectively, and the median RFS and median OS time in FLT3-group had not yet reached. There were no significant differences of all the indexes between the two groups (all P< 0.01). And there were no significant differences in CR rate, 2-year RFS rate and 2-year OS rate between NPM1+group and NPM1-group (all P＞0.05). The CR rate, 2-year RFS rate and 2-year OS rate in NPM1+ FLT3-group were better than those in NPM1- FLT3-, NPM1-FLT3+and NPM1+FLT3+groups; NPM1-FLT3+and NPM1+FLT3+groups had the worst prognosis, and there were no statistical differences in the CR rate, RFS and OS time between the two groups (all P＞ 0.05). The prognosis of the patients in bone marrow juvenile cell ratio < 0.05 group at T time point was better than that in ratio ≥0.05 group (all P< 0.05). CN-AML patients were classified into the good prognosis group (NPM1-FLT3-), the medium prognosis group (NPM1+FLT3-), and the poor prognosis group (NPM1-FLT3+and NPM1+FLT3+) according to the FLT3 and NPM1 genes. The good prognosis group+the ratio of bone marrow juvenile cells at T time point<0.05 group, the good prognosis group+the ratio of bone marrow juvenile cells at T time point≥0.05 group, the medium prognosis group + the ratio of bone marrow juvenile cells at T time point < 0.05 group had no statistical differences in CR rate, 2-year RFS rate and 2-year OS rate (all P ＞0.05). The medium prognosis group + the ratio of bone marrow juvenile cells at T time point ≥0.05 group, the poor prognosis group+the ratio of bone marrow juvenile cells at T time point<0.05 group had the equivalent prognosis, and the average prognosis was moderate; the poor prognosis group + the ratio of bone marrow juvenile cells at T time point ≥ 0.05 group had the worst prognosis. According to Cox multivariate regression analysis, FLT3 gene mutation and the ratio of bone marrow juvenile cells at T time point were independent influencing factors for RFS and OS in CN-AML patients (all P< 0.05). NPM1 was an independent prognosis factor affecting RFS and OS of FLT3-patients (all P < 0.05). Conclusions After induction chemotherapy, the responsiveness and sensitivity of AML patients to chemotherapy regimen can be assessed early and objectively according to molecular genetics and the ratio of bone marrow juvenile cells at T time point, which has a certain value in the prognosis judgement.

Reclassification of Acute Myeloid Leukemia According to the 2016 WHO Classification

We reviewed our leukemia database to reclassify 610 patients previously diagnosed as having acute myeloid leukemia (AML) according to the updated 2016 WHO classification. Nine patients were categorized as having myelodysplastic syndrome and myeloid neoplasms with germline predisposition. AML with recurrent genetic abnormalities accounted for 57.4% (345/601) of the patients under the 2016 WHO classification. AML with mutated NPM1 was the most common form (16.5%), with the majority associated with monocytic differentiation (63.6%). AML with double CEBPA mutations accounted for 8.3% of these cases, and the majority were previously diagnosed as AML with/without maturation (78.0%). These newly classified mutations were mutually exclusive without overlapping with other forms of AML with recurrent genetic abnormalities. AML with mutated NPM1 and AML with myelodysplasia-related changes comprised the oldest patients, whereas AML with RUNX1-RUNX1T1 included the youngest patients. The leukocyte count was highest in AML with mutated NPM1, and the percentage of peripheral blood blasts was the highest in AML with double CEBPA mutations. Our results indicate that implementation of the 2016 WHO classification of AML would not pose major difficulties in clinical practice. Hematopathologists should review and prepare genetic tests for the new classification, according to their clinical laboratory conditions.

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.

Study of the effects and mechanism of all-trans retinoic acid on leukemic cell line U937 cells with NPM1 mutation / 中华血液学杂志

Objective@#To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation.@*Methods@#Human acute myeloid leukemia cell line U937 was explored, NPM1 mutated (A type) plasmids were transfected into U937 to form stable clones A1 and A2, which were identified by Western blot and Co-immunoprecipitation. The cell proliferation was measured by methylthiazolyl tetrazolium bromide (MTT) ; cell cycle and cell apoptosis were explored by flow cytometric; cell colony formation was measured by microscope count, the molecular pathways related to cell proliferation were measured by Western blot.@*Results@#①The cell proliferations of mutant A1 and A2 were inhibited significantly by 52.6% and 35.8% (P<0.05) , respectively under ATRA exposure. ②The percentages of G0/G1 stage of mutant A1 and A2 increased by 20.1% and 35.8%, respectively under ATRA exposure. ③All the U937 leukemic cells were inhibited under ATRA exposure; the decreased percentages of vector, wild-type and mutant NPM1 cells were 32.7%, 57.9% and 90.9% respectively. ④p-ERK decreased obviously after ATRA exposure in NPM1 mutated leukemic cells. ⑤More mutant NPM1 cells inclined to apoptosis under the exposure of ATRA and cytotoxic drugs than cytotoxic drugs alone, meanwhile more cells apoptosis occurred when ATRA was administrated after cytotoxic drugs exposure.@*Conclusions@#ATRA could inhibit cell proliferation and colony formation, blocked the cell cycle in the G0/G1 stage accompanied by the significant reduction of p-ERK in U937 leukemic cells with NPM1 mutation. Besides, ATRA could synergize with drugs to suppress the leukemic cells survival more effectively when ATRA was administered after the cytotoxic drugs exposure in U937 leukemic cells with NPM1 mutation.

Prognostic significance of early assessment of minimal residual disease in acute myeloid leukemia with mutated NPM1 patients / 中华血液学杂志

Objective@#To explore prognostic significance of early assessment of minimal residual leukemia (MRD) in adult patients with de novo acute myeloid leukemia (AML) with mutated NPM1.@*Methods@#The response, NPM1 mutated transcript level after induction chemotherapy and the first 2 cycles of consolidation chemotherapy, disease-free survival (DFS) and overall survival (OS) in 137 patients with AML with NPM1 mutations of A, B and D were retrospectively analyzed.@*Results@#Data of 137 patients were collected, 67 were male, the median age was 49 years (16-67 years) , 107 (78.1%) had normal karyotype, 57 (41.6%) had positive FLT3-ITD mutation, the median NPM1 mutated transcript level at diagnosis was 84.1%. Among the 134 evaluable patients, 115 (85.8%) achieved a complete remission (CR) . Multivariate analyses revealed that WBC<100×109/L (OR=0.3, 95% CI 0.1-0.9, P=0.027) and first induction therapy with "IA10" protocol (OR=0.3, 95% CI 0.1-0.8, P=0.015) were factors associated with achieving a CR. With a median follow-up period of 24 months (range, 2 to 91 months) in 77 survived CR patients, the probabilities of DFS and OS at 3 years were 48.0% and 63.9%, respectively. Multivariate analyses showed that positive FLT3-ITD (HR=3.2, 95% CI 1.6-6.7, P=0.002) , high MRD level after 2 cycles of consolidation chemotherapy (NPM1 mutation transcript level <3-log reduction from the individual baseline, HR=23.2, 95% CI 7.0-76.6, P<0.001) and chemotherapy or autologous hematopoietic stem cell transplantation (auto-HSCT) rather than allogeneic HSCT (allo-HSCT) (HR=2.6, 95% CI 1.0-6.6, P=0.045) were the unfavorable factors affecting DFS, high MRD level at the time of achieving the first CR (NPM1 mutation transcript level <2-log reduction from the individual baseline, OR=2.5, 95% CI 1.0-6.1, P=0.040) and after 2 cycles of consolidation chemotherapy (HR=4.5, 95% CI 2.0-10.3, P<0.001) were the unfavorable factors affecting OS. Furthermore, DFS and OS rates at 3 years in those receiving chemotherapy or auto-HSCT were 39.7% and 59.1%, respectively; positive FLT3-ITD and high MRD level after 2 cycles of consolidation chemotherapy were independent factors associated with both shorter DFS (HR=3.5, 95% CI 1.6-7.6, P=0.002 and HR=8.9, 95% CI 3.8-20.7, P<0.001) and OS (HR=2.7, 95% CI 1.1-6.9, P=0.036 and HR=3.1, 95% CI 1.2-8.0, P=0.021) ; meanwhile, high MRD level at the time of achieving the first CR associated with shorter OS (HR=3.1, 95% CI 1.2-8.0, P=0.022) .@*Conclusion@#Positive FLT3-ITD mutation and high MRD level after induction or consolidation chemotherapy associated with poor outcomes in AML patients with mutated NPM1.

Effects of NPM1 gene expression on acute myeloid leukemia cell lines and its mechanism / 中华血液学杂志

Objective@#To investigate the impact and mechanism of NPM1 gene expression on acute myeloid leukemia (AML) cell lines.@*Methods@#Human AML cell line U937 and HL-60 cells were transfected with NPM1 plasmid to establish stable clones, and the high NPM1 protein expression (NPM1hi) clones were screened by Western blot. The cell proliferation was assayed by methylthiazolyl tetrazolium bromide (MTT) , cell cycle and cell apoptosis by flow cytometric, cell colony formation by microscope count, the molecular pathways related to cell cycle by Western blot. The expression of NPM1 gene in primary AML bone marrow mononuclear cells (BMMC) was investigated by RQ-PCR.@*Results@#①The proliferation of NPM1hi U937 and HL-60 cells was similar with that of control cells (4.68±1.28 vs 3.89±0.81, 3.34±0.37 vs 2.68±0.29, P>0.05) . ②The percentage of S phase in NPM1hi U937 and HL-60 cells was higher than that in control cells[ (50.22±3.42) % vs (39.78±3.80) %, (59.01±3.27) % vs (43.94±2.08) %, P<0.05]. ③The anti-apoptosis capacity and colony formation abilities of NPM1hi U937 cells increased than that of control cells[ (68.8±10.2) % vs (48.7±3.22) %, and (772.7±24.0) vs (652.3±16.5) , P<0.05], but the above abilities of NPM1hi HL60 cells were similar with that of control cells. ④ The expressions of CDK4, Cyclin D1, Cyclin D2 and Cyclin E in NPM1hi leukemia cells were higher than that of control cells, but the expression of Cyclin D3 was lower. ⑤The NPM1 expression levels in AML patients with favorable cytogenetic prognosis were lower than that of patients with intermediate prognosis.@*Conclusions@#NPM1 protein could promote more cells to enter S phase, enhance the ability of antiapoptosis and colony formation in AML cell lines. The quantitative level of NPM1 may predict the cytogenetic risk of AML patients.

Frequency and Distinct Characteristics of Acute Myeloid Leukemia Lacking HLA-DR and CD34 Expression: Features Intermediate between Typical Acute Myeloid Leukemia and Acute Promyelocytic Leukemia

BACKGROUND: The objective of this study was to investigate the frequency and characteristics of HLA-DR⁻/CD34⁻ acute myeloid leukemia (AML) also known as acute promyelocytic leukemia (APL)-like AML. METHODS: This study included 683 newly diagnosed patients with AML. After exclusion of 211 patients with recurrent genetic abnormalities, one with acute panmyelosis with myelofibrosis, two with myeloid leukemia associated with Down syndrome, and two devoid of metaphase cells, we classified the remaining 467 patients as follows: group 1, HLA-DR⁺/CD34⁺ (typical AML); group 2, HLA-DR⁺/CD34⁻ or HLA-DR⁻/CD34⁺; group 3, APL-like AML. RESULTS: Group 1 comprised 294 patients, group 2 comprised 133, and group 3 comprised 40. Therefore, the frequency of APL-like AML among 683 unselected patients with AML was 5.9%. Group 3 patients had significantly higher leukocyte counts and bone marrow (BM) blast percentages, higher frequencies of normal karyotypes and NPM1 mutation, higher fractions of CD33-positive cells, higher concentrations of fibrin degradation products and D-dimers, lower frequencies of complex karyotypes, monosomal karyotypes and poor cytogenetic risk, lower fractions of CD13-positive cells, and lower fibrinogen concentrations, compared with group 1 patients. The values of the BM blast percentage, number of CD33-positive cells, and DIC score of the patients with APL-like AML were intermediate between those of the patients with typical AML and APL. CONCLUSIONS: This study demonstrates that APL-like AML is not uncommon, and it has characteristics distinguishable from those of typical AML. APL-like AML may have some pathophysiological relationships with APL, which need further investigation.

Nucleophosmin mutation analysis in acute myeloid leukaemia: Immunohistochemistry as a surrogate for molecular techniques.

Background & objectives: Mutation of nucleophosmin (NPM1) gene in the absence of FLT3-ITD (FMS related tyrosine kinase 3 - internal tandem duplications) mutation carries a good prognosis in cytogenetically normal acute myeloid leukaemia (AML). NPM1, a multifunctional nucleolar phosphoprotein that shuttles between nucleus and cytoplasm, gets trapped in the cytoplasm when mutated. Immunohistochemical (IHC) demonstration of its aberrant cytoplasmic location (NPMc+) has been suggested as a simple substitute for the standard screening molecular method. This study was aimed to assess the diagnostic utility of IHC on formalin fixed bone marrow biopsies in comparison with the reference molecular method (allele specific oligonucleotide - polymerase chain reaction; ASO-PCR) to predict NPM1 mutation status in AML patients. Methods: NPM protein IHC was performed using mouse anti-NPM monoclonal antibody on 35 paraffin-embedded bone marrow biopsies of patients with primary AML of any French-American-British (FAB) subtype. Results of IHC were compared with those of ASO-PCR. Results: Of the 35 AML patients, 21 (60%) were positive for NPM1 exon 12 gene mutation by ASO-PCR, 19 (90.47%) of these 21 were NPMc+. Thirteen of the 35 patients were negative by both the methods. One NPMc+ patient was not detected by ASO-PCR. IHC had a sensitivity and specificity of 90 and 93 per cent, respectively, compared to the molecular screening gold standard. Interpretation & conclusions: Mutation of NPM1 determined by the widely available and inexpensive IHC agrees closely with results of the standard molecular methods. Thus, technically and financially not well endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC.

Detection and clinical research of FMS-like tyrosine kinase-3, NPM1 and c-kit genes in bone marrow slides of patients with acute myeloid leukemia / 白血病·淋巴瘤

Objective To study the FMS-like tyrosine kinase-3 (FLT3) gene, NPM1 gene and c-kit gene mutations in acute myeloid leukemia (AML) by extracting DNA from the storage of bone marrow slides, and to investigate the relationship between the three gene mutations and clinical features in AML. Methods The bone marrow slides of 55 patients diagnosed with AML were enrolled in this study. The PCR, DNA sequencing and molecular cloning were used to detect and analyse the FLT3-ITD, NPM1 and c-kit gene mutations. Patients' remission, progression and survival time were also recorded. Results The DNA was successfully extracted from the bone marrow slides with -20 ℃ frozen storage without Wright stained, chemically fixed, and room temperature storage Wright stained discoloured by phenol ∶ chloroform ∶ isoamyl alcohol method, which can be used in PCR, direct sequencing and molecular cloning sequencing analysis. 10 of the 55 cases (18.2 %) were FLT3-ITD positive, including 9 cases with heterozygous mutations and 1 case with homozygous mutation. FLT3-ITD positive group had lower complete remission (CR) rate, shorter event-free survival (EFS) time and overall survival (OS) time than the negative group (P< 0.05). 9 of the 55 cases (16.4 %) had NPM1 heterozygous gene mutations, all belonging to type A. The EFS rate of the patients with NPM1 mutation was higher in 10 months and the OS rate was higher in 19 months (P< 0.05). 3 of 9 NPM1 mutations patients were FLT3-ITD positive. The CR rates of the four groups after initial remission induction therapy in order were NPM1+FLT3-ITD-, NPM1-FLT3-ITD-, NPM1-FLT3-ITD+, NPM1+FLT3-ITD+(P<0.05). Besides, NPM1-FLT3-ITD+was a risk factor affecting the OS (RR=1.250, P=0.005). 2 of the 55 cases (3.6 %) had c-kit gene mutations, namely mutant D816H and mutant D816V. The c-kit gene mutations were not found in patients with FLT3-ITD and NPM1 mutations. Conclusions The FLT3-ITD mutation is a poor prognosis molecular marker in AML, and NPM1 mutation is a good factor for the prognosis. NPM1-FLT3-ITD+is a risk factor affecting OS. The rate of c-kit gene mutation is low in AML, without the overlap of FLT3 and NPM1 mutations.