ABSTRACTand clinical impact of these mutations in the context of other clinical and genetic factors in a well-defined population of patients intensively treated in trials of the GermanAustrian AML Study Group (AMLSG).
Methods
PatientsA total of 1696 younger AML patients (18 to 61 years) were studied. Patients were enrolled in prospective treatment protocols of the AMLSG), namely AML HD98A 12 (n=733; NCT00146120), AMLSG 07-04 13 (n=893; NCT00151242), and APL HD95 14 (n=70) for the patients with acute promyelocytic leukemia (APL). The clinical studies were approved by the local ethics review committees and all patients gave informed consent for both treatment and cryopreservation of leukemia samples according to the Declaration of Helsinki. The only criterion to include patients in our study was the availability of a pretreatment bone marrow or peripheral blood specimen for analysis of ASXL1 mutations. Cytogenetic and additional molecular analyses were performed as previously described.
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ASXL1 mutation analysisA detailed description of the ASXL1 mutation analysis is provided in the Online Supplementary Material. Briefly, genomic DNA was used as a template for polymerase chain reactions to amplify several fluorescently-labeled DNA fragments covering the entire exon 12 (AMLSG 07-04) or regions within exon 12 (AML HD98A and APL HD95) identified as main mutation clusters in AML. 6,20 Amplicons were screened for mutations by a GeneScan-based fragment analysis (Online Supplementary Figures S1 and S2). Samples classified as mutated after the GeneScan analysis (Online Supplementary Figure S2) were further analyzed by direct sequencing to validate the mutation and to determine the mutation type.
Statistical analysisStatistical analyses of clinical outcome were performed according to previous reports. 16 The median follow-up for survival was calculated according to the method of Korn. 21 The definition of complete remission (CR), event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS) as well as cytogenetic categorization into favorable-, intermediate-, and adverse-risk groups followed recommended criteria. 22 Pairwise comparisons between patients' characteristics (covariates) were performed using the Mann-Whitney test for continuous variables and the Fisher exact test for categorical variables. The Kaplan-Meier method was used to estimate the distribution of EFS, RFS and OS. 23 Estimation of confidence intervals for the survival curves was based on the Greenwood formula for standard error estimation. A logistic regression model was used to analyze associations between baseline characteristics and the achievement of CR. A Cox model was used to identify prognostic variables. 24 In addition to ASXL1 mutation status, age, sex, hemoglobin level, logarithm of white blood cell count, type of AML (de novo, secondary AML, therapy-related AML), percentage of peripheral blood and bone marrow blasts, cytogenetic risk group, 22 and mutational status of NPM1, FLT3 (ITD and TKD),), RUNX1, MLL (PTD), and DN...