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.

OBJECTIVE:To analyze the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) detected by next generation sequencing (NGS) and their coexistence and mutual exclusivity relationship in the AML subtype.METHODS:The NGS data based on 112 genes related to blood disease in 238 newly diagnosed patients with NPM1mut were collected. The χ2 test and non-parametric test were used to analyze the distribution correlation between the genes in the mutational spectrum.RESULTS:Among all the patients, at least one co-mutation was detected out. The median number per case of the mutated genes, including NPM1mut was 4.5 (range 2－14), among them, there were 5.0 (range 2－10) for NPM1mut/FLT3-ITD+ and 4.0 (range 2－14) for NPM1mut/FLT3-ITD- cases, but it was no significant difference between the two groups (P=0.378). A total of 240 NPM1 mutational events were detected out in entire 238 NPM1mut patients, of which 10 (4.2%) were missense mutations, and were all found in NPM1mut/FLT3-ITD- patients. Most (9/10, 90%) of these NPM1 missense mutations were accompanied by AML subtype-defining cytogenetic or molecular abnormalities, of which 7 patients were in low risk or 2 in high risk. The most common NPM1mut coexisting mutations were DNMT3A (104, 43.7%), followed were FLT3-ITD (95, 39.9%) and FAT1 (57, 23.9%), FLT3-ITD and DNMT3A showed significant coexistence (P=0.005). FLT3-ITD showed significantly reciprocal exclusivity with FLT3-nonITD (P<0.001), NRAS (P<0.001), PTPN11 (P=0.017) and IDH1 (P=0.005), and showed an exclusivity inclination with KRAS (P=0.073). In addition, FLT3-nonITD along with KRAS (P=0.035), NRAS along with KRAS (P=0.008) and PTPN11 (P=0.039) coexisted significantly.CONCLUSION:Prognoses of AML involving less common NPM1 missense mutations should be stated on a case by case basis. The mutational landscape and co-occurrence and mutual exclusivity correlations of NPM1mut AML provide a mechanism explaining biological diversity and clinical heterogeneity in this AML subset.

OBJECTIVE To explore the effect of miR-100 on the migration of rat bone marrow mesenchymal stem cells(rBMMSC). METHODS The rBMMSC were isolated by density gradient centrifugation, cell surface epitopes of CD105, CD45, CD34, CD29 and CD44 were analyzed by flow cytometry. The rBMMSC were transfected with miR-100 mimic or inhibitor, then the expression of miR-100 in transfected cells was detected by real-time PCR. Migration test was used to observe the effect of miR-100 on cell migration ability. The secretion level of chemokine SDF-1 in culture supernatant of cells was quantitatively detected by ELISA. RESULTS The isolated cells were identified as BMMSC. After rBMMSC were transfected with miR-100 mimic or inhibitor, as compared with control group,the expression of miR-100 in rBMMSC significantly increased or decreased respectively. In the migration experiment, the rBMMSC migration was significantly inhibited in the miR-100 mimic group (P<0.01), while the rBMMSC migration was significantly enhanced in the miR-100 inhibitor group (P<0.01). The concentration of SDF-1 in the supernatant of the miR-100 mimic group and the miR-100 inhibitor group did not change significantly compared with the control group (P>0.05). CONCLUSION miR-100 can significantly inhibit the migration of rBMMSC, but not significantly correlated with the SDF-1.

Objective To analyze the heterogeneity of remission and prognosis of patients with t(8; 21) acute myeloid leukemia (AML) under current treatment modalities, and to provide a basis for further risk-adapted treatment. Methods A total of 107 adult patients with primary t(8; 21) AML treated with the standard 3+ 7 regimen at Changzhou First People's Hospital from October 2014 to September 2018 were collected. The complete remission (CR) rate, cumulative incidence of relapse, event-free survival (EFS), and overall survival (OS) were evaluated by combining the characteristics of traditional Morphology, Immunology, Cytogenetics, and Molecular biology (MICM) and genetic mutations based on next-generation sequencing (NGS) using multivariate Logistic and Cox regression analyses. Results After a single induction course, the CR rate was 79.0% (83/105) and the early mortality rate was 1.9% (2/107). Multivariate analysis showed that positive KIT-D816mut was the only independent factor adversely affecting the CR rate (hazard ratio [HR]=3.29 [1.18-9.24], P=0.023), EFS (HR=3.53 [1.82-6.84], P=0.000), and OS (HR=5.45 [1.77-16.84], P=0.003). Negative CD19 expression was the only independent predictor of increased cumulative incidence of relapse (HR=0.32 [0.10-1.00], P=0.050). KITmut, KIT-N822, and complex karyotype were not independent risk factors for all endpoints. Conclusions It is suggested that the specific KIT-D816mut, rather than general KITmut, should be included in the risk stratification system of t(8; 21) AML. Patients with negative CD19 have an increased risk of relapse, which requires close monitoring. Key words: t(8; 21); Acute myeloid leukemia; CD19; Next-generation sequencing; KIT; Prognosis

Introduction Cytogenetics and molecular testings for disease classifying and prognosis estimation are becoming routine in clinical practice. However, the molecular characteristics of acute monocytic leukemia (AML-M5) remain unclear. The aim of this study was to investigate the association between karyotypes and gene mutations, especially in AML-M5 patients with 11q23/KMT2A (MLL) rearrangement and normal karyotype. Methods A total of 126 de novo AML-M5 patients were screened for mutations in the 51 genes known or suspected to have a role in myeloid malignancies or in monocytic differentiation using next-generation sequencing (NGS). Chromosome karyotype analysis was performed by R-banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT-PCR). Results Of the 126 patients, one or more mutations were detected in 83.3% patients. FLT3-ITD and NRAS had the highest mutation frequency, followed by NPM1, DNMT3A, TET2, KRAS, and RUNX1. We also identified a significant difference in mutational spectrums between KMT2A-rearranged (KMT2Ar) patients and normal karyotype (NK) patients, as reflected in the average number of gene mutations per patient (1.66 vs 2.46), and in the frequencies of commonly mutated genes (FLT3-ITD: 6% vs 43.5%; NPM1: 0% vs 43.5%; RUNX1: 2.0% vs 15.2%; DNMT3A: 4% vs 26.1%; KRAS: 24.0% vs 4.35%). Patients harboring >= 3 mutations showed much lower complete remission rate than that with double mutations (P = 0.043) in high-risk group. Conclusion There was a significantly different mutation profile between KMT2Ar-patients and NK patients. Our research provided new insight into the molecular characteristics of AML-M5.

目的:探讨miR-378对异体移植过程中骨髓间充质干细胞(BMSCs)存活的影响.方法:采用密度梯度离心法分离大鼠BMSCs(rBMSCs),流式细胞仪检测rBMSCs表面标志物CD105、CD45、CD34、CD29和CD44的表达情况,茜素红和碱性磷酸酶染色观察细胞成骨诱导分化能力.采用miR-378模拟物或抑制剂转染rBMSCs,Real-time PCR检测细胞中miR-378表达情况.使用大鼠异体血清(ARS)干预转染后的rBMSCs,采用甲基偶氮唑蓝(MTT)法检测细胞存活率,AnnexinⅤ/PI双染流式细胞术检测细胞凋亡,Western blot检测凋亡蛋白Caspase-3表达水平.Europium标记转染后rBMSCs进行大鼠尾静脉移植,观察活体内rBMSCs存活情况.结果:经鉴定所分离的细胞确定为rBMSCs.转染miR-378模拟物(或抑制剂)后,rBMSCs中miR-378的表达显著增加(或降低)(P<0.05).ARS干预后,rBMSCs存活率显著降低(P<0.05),凋亡率显著升高(P<0.05),cleaved-caspase-3表达显著上调(P<0.05);miR-378过表达(或低表达)可显著提高(或降低)ARS干预下rBMSCs的存活率,降低(或增加)细胞凋亡率,下调(或上调)cleaved-caspase-3表达.尾静脉异体移植rBMSCs后,miR-378过表达(或低表达)可显著提高(或降低)rBMSCs在活体内的存活率(P<0.05).结论:miR-378可显著提高异体移植过程中BMSCs的存活率,且抑制细胞凋亡.