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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.
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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.
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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.
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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.
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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.
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Objective Toinvestigatetheimmunophenotypiccharacteristicsofacutemyeloidleukemia(AML) patients with NPM1 mutation. Methods The immunophenotype of 237 newly diagnosed AML patients were detected by flow cytometry. Real-time quantitative PCR was employed to detect the NPM1 mutation. The immunophenotype was then compared between the NPM1 mutated and wild type patients. Results The incidence of NPM1 mutation was 19.0 ％ (45/237) in all AML patients.The NPM1 mutated patients had lower expression of CD34,CD117,HLA-DR,CD15 and CD19 than the wild type patients(all P＜0.05).For AML patients with normal karyotype,the incidence of NPM1 mutation was 37.7 ％ (40/106),and the NPM1 mutated patients had lower expression of CD34,HLA-DR,CD15 and CD7 than the wild type patients(all P＜0.05).The NPM1 mutated patients with normal karyotype had lower expression of CD34 HLA-DR and CD7 in M1 subtype(all P ＜ 0.05); lower expression of HLA-DR and higher expression of CD9 in M2 subtype (all P ＜ 0.05) ; and lower expression of CD117 in M5 subtype compared with wild type patients (P ＜0.05). Conclusion The immunophenotypic characteristics of AML patients are changed by NPM1 mutation. The changes of immunophenotype varied in different FAB subtypes.