|
[Objective]1. To analyze cytogenetic characters on4241consecutive and unselected de novo AML patients from a single center in China and compare our results with that of other Asian and western countries2. To investigated the prevalence of16gene mutations including C-KIT, NPM1, FLT3-TKD, FLT3-ITD, MLL-PTD, EZH2, RUNX1, ASXL1, IDH1, IDH2, NRAS, CBL, WT1, TET2, DNMT3A and JAK2on the molecular level by sequencing coding exons in a cohort of430de novo AML patients, analyzed the relationship with clinical features, MICM characters and risk-status categories, evaluated the correlation among gene mutations and the prognostic relevance with treatment.3. To determine the incidence and clinical implications of somatic EZH2mutations in714patients with de novo AML.[Methods]1. From April1985to November2011, samples from4196newly diagnosed patients with de novo AML were received at the cytogenetic laboratory of the First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology for cytogenetic analysis. R-banded karyotypic analyses were then performed according to standard procedures of our laboratory.Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature (ISCN2009).2. Genomic DNA of all430patients was extracted from frozen bone marrow mononuclear cells (BMMCs) after Ficoll gradient centrifugation using standard procedures. Mutations of C-KIT, NPM1, FLT3-TKD, FLT3-ITD, MLL-PTD,EZH2, RUNX1, ASXL1, IDH1, IDH2, NRAS, CBL, WT1, TET2, DNMT3and JAK2were analyzed by PCR amplification followed by direct DNA sequencing. The clinical and lab data of these cases were collected, and their clinical characteristics therapies and survival prognosis were analyzed.3. EZH2mutations were analyzed by PCR amplification of the entire coding region of PHF6exons2-10followed by direct bidirectional DNA sequencing in714AML patients from January2005to December2010.[Results]1. Cytogenetic characters on4241de novo AML patients from a single center Karyotypic analyses were successfully performed in4196patients (98.9%) at diagnosis. Chromosome abnormalities were detected in2405patients (57.3%). Besides the aberrations previously reported in the literature,65novel translocations were observed in this study. t(15;17) represented the most frequent anomaly(19.3%) in our cohort, followed by t(8;21)(14.3%), complex (5.3%),+8(3.2%). The percentage of t(15;17) and t(8;21) was comparable with that in most East Asian countries, which was obviously higher than reported in Central-North Europe and the United States. Among the FAB subtype, the frequency of M3was higher while M4was lower compared with that of western countries.2. Gene mutation patterns77.2.0%of430AML patients and84.3%of178normal karyotype AMLs were found to have at least one mutation. RUNX1and IDH1mutation had the tendency to occur in the old age (P=0.004, P=0.038) and MLL-PTD mutation exclusively in male patients12/14(85.7%). NPM1, FLT3-ITD, DNMT3A mutations were significantly associated to high WBC count (30.0×109/L)(P<0.0001, P<0.0001and P=0.021respectively), MLL-PTD and EZH2to less percentage of blasts in bone marrow(<30%)(P<0.0001and p=0.023) and MLL-PTD, RUNX1mostly to BM immunophenotype of both myeloid and monocytic (P<0.0001and p=0.015). C-KIT mutation overwhelming occurred in core binding factor (CBF) leukemias (73.5%)(P<0.0001), while NPM1(81.1%), IDH2(63.8%) and DNMT3A (85.2%) mostly occurred in normal karyotype AMLs.NPM1mutation often overlapped with the others genes, associated with FLT3-ITD (37/99, P<0.0001), IDH1(13/20, P<0.0001), IDH2(24/47, P<0.0001), TET2(17/47, P=0.007) and DNMT3A (18/27, P<0.0001).Prognosis analysis showed FLT3-ITD, IDH1, NRAS mutations were associated with a significant lower CR rate (P=0.007,0.031and0.031, respectively), FLT3-ITD, NRAS and risk-status subgroup were independent influence factors for CR rate (P<0.0001, p=0.003, P=0.006respectively). By Kaplan-Meier survival analysis, NPM1, FLT3-ITD, MLL-PTD, RUNX1, IDH1, IDH2, NRAS and DNMT3A mutations predicted the poor EFS and OS(EFS:P=0.0148, P=0.0164, P=0.0018, P=0.0015, P=0.0213, P=0.0116, P=0.0001and P<0.0001, respectively and OS:P=0.0077,P=0.0039, P=0.0177, P=0.0150, P=0.0014, P=0.0159, P<0.0001, P=0.0001, respectively).In a multivariate Cox regression model, mutations of FLT3-ITD, NRAS, IDH2, age and risk-status category were revealed independent prognostic significance (EFS: P<0.0001,0.029,0.046,<0.0001<0.0001;OS:<0.0001,0.001,0.005,<0.0001,<0.0001).3. Prevalence and prognostic value of somatic EZH2mutations in714patients with de novo AML.EZH2mutations were identified in13/714(1.8%) of AML patients and occurred more in males (P=0.033). The presence of EZH2mutations was significantly associated with lower blast percentage (21-30%) in bone marrow (P<0.0001) and-7/del(7q)(P=0.025). There were no differences in the incidence of mutations in13genes, including ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2mutations.Because of rarity of EZH2mutations in de novo AML, the prognostic impact of EZH2mutations in AML is still uncertain, and will need to be assessed in larger cohorts of patients collected on multi-center co-operative studies, though there were no significant difference on EFS and OS between EZH2mutated patients and wild-type in this study.[Conclusions]1. Age at diagnosis of AML was much younger than those from the western countries. AML patients of the East Asian population may predispose to have a favorable karyotype and the M3subtype.2. Gene mutations were demonstrated related with clinical features and valuable prognostic markers of AML.3. Somatic mutations of EZH2may play an important role in pathogenesis of de novo AML patients with-7/del(7q) or with21-30%of blasts in BM.The prognostic impact of EZH2mutations in AML is still uncertain, though there were no significant difference on EFS and OS between EZH2mutated patients and wild-type in this study. 4. To evaluate exactly prognosis risk status and formulate the personal therapeutic regimen, synthetic analysis of all the factors including age, physical condition, clinical feathers, and MICM characters, especially in cytogenetics and genetic mutation pattern, should extremely been taken into account in advanced
|