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V.V. Prassek et al.
can result in prolonged overall survival (OS), at least in a subset of elderly patients aged 70-79 years, and may even be beneficial in selected octogenarians.4,7-10 However, it remains unclear which genetic and clinical factors are rel- evant to identify those elderly patients most likely to ben- efit from, and least likely to be harmed by, induction chemotherapy.11
Advances in the field of molecular genetics and the development of next-generation sequencing expanded our knowledge of recurrently mutated genes in AML and their role in disease pathophysiology, and led to refined risk classifications in younger patients.1,12,13 However, elderly patients were underrepresented or excluded in these stud- ies, and the question thus arises whether the spectrum and prognostic relevance of gene mutations are similar in very old AML patients. Some studies of established genet- ic risk factors indicate that there may be important differ- ences between elderly and younger patients.12,14,15 For example, the FLT3-ITD mutation is a well-recognized adverse prognostic factor in young adults, whereas its impact is reduced or absent in elderly patients.1,7,14,16,17 Therefore, comprehensive genetic analyses in cohorts of very old patients are needed to clarify the relevance of dis- tinct gene alterations in this subset of patients.
To identify prognostic factors associated with clinical outcomes in elderly AML patients, we studied 151 patients aged ≥75 years who received intensive induction therapy. The mutational spectrum in 64 recurrently mutat- ed AML genes was analyzed by targeted next-generation sequencing. We then studied associations of genetic alter- ations with other known prognostic factors, patients’ characteristics, and outcomes. Our aim was to define sub- sets of patients who may benefit from intensive induction therapy. Furthermore, we evaluated the prognostic rele- vance of the British Medical Research Council (MRC) 2010 and European LeukemiaNet (ELN) 2017 risk classifi- cation schemes in this subset of patients.1,18
Methods
previously.12 The median sequencing coverage of the target region across all samples was 520-fold, and 98.8% of the target region was covered at >30-fold. Gene alterations with variant allele fre- quencies of ≥2% were classified as driver mutations, variants of unknown significance or germline polymorphisms.12 The Catalogue Of Somatic Mutations In Cancer (COSMIC), The Cancer Genome Atlas (TCGA) and the Single Nucleotide database (dbSNP) served as databases to compare and classify the muta- tional results.19-21 NPM1,22 FLT3-ITD23 and CEBPA24 mutations were additionally tested using polymerase chain reaction followed by Sanger sequencing and/or fragment analysis.
Written informed consent for inclusion in the clinical trial and genetic analyses was provided by all patients. All study protocols were in accordance with the Declaration of Helsinki and approved by the institutional review boards of each participating center.
Statistical analysis
Associations among gene mutations, and between gene muta- tions and patients’ pretreatment features, were analyzed using the Fisher exact test for categorical variables and the Wilcoxon rank- sum test for continuous variables. The Kaplan-Meier method was used to calculate estimated survival probabilities, with the log- rank test evaluating differences between survival distributions. A multivariate Cox regression model including known risk factors [MRC risk category and Eastern Cooperative Oncology Group performance status (ECOG PS)], and gene mutations that showed a univariate association with OS (at P<0.10), was used to identify factors associated with survival. Statistical analyses were per- formed using SPSS version 24.0 (Chicago, IL, USA). All statistical tests are two-sided, and a P value of ≤0.05 was considered statis- tically significant.
Results
Patients’ characteristics
Patients, treatment, karyotype and molecular analyses
We identified 151 patients aged ≥75 years treated in the AMLCG-1999 trial for whom suitable material for genetic analyses was available. Details of this trial have been pub- lished elsewhere.25 The patients’ baseline characteristics are shown in Table 1. The median age of the subjects was 76 years (range, 75-86). Eighty-one percent of patients had a clinical diagnosis of de novo AML, 15% had secondary AML and 3% had therapy-related AML. Three patients (2%) had high-risk myelodysplastic syndromes (10-<20% bone marrow blasts). Most patients (90%) had an ECOG PS of ≤2. Among 137 patients with cytogenetic data, 82% belonged to the intermediate-risk group according to the MRC 2010 classification, including 52% with cytogeneti- cally normal AML. Only three patients (2%) had favorable cytogenetics, and 16% fell in the MRC adverse-risk group.
We studied 151 patients aged ≥75 years with newly diagnosed AML or high-risk myelodysplastic syndromes (10% - <20% bone marrow blasts; n=3), diagnosed according to World Health Organization criteria, who had suitable bone marrow or peripher- al blood specimens for genetic analysis. All patients received inten- sive induction treatment during the German AML Cooperative Group AMLCG-1999 randomized, multicenter, phase III trial (clin- icaltrials.gov identifier, NCT00266136) between 1999 and 2011. Participants aged ≥60 years were randomized to receive a first induction course with high-dose cytarabine and mitoxantrone (HAM) or with standard-dose cytarabine, daunorubicin and 6- thioguanine (TAD-9). A second HAM induction course was administered from day 21 only if ≥5% residual blasts were present in a bone marrow aspirate taken on day 15. Another cycle of TAD-9 was given as consolidation therapy, followed by monthly cytarabine-based maintenance chemotherapy (Online Supplementary Data). Karyotype analyses were performed central- ly and results were classified according to the 2010 MRC classifi- cation.18
Treatment outcomes
In the overall cohort, the rates of complete remission (CR) and CR with incomplete blood count recovery (CRi) were 44% and 4%, respectively. The median event-free survival (EFS) was 1.7 months. The median relapse-free survival (RFS) for patients achieving a remission was 12 months, and the median OS was 6.0 months (Online Supplementary Figure S1). The 3-year OS rate for the entire study population was 21%, and survival was similar for patients aged 75-79 or 80-86 years (P=0.3) (Online Supplementary Figure S2A). Patients with an ECOG PS of 3 or 4 had shorter OS compared to patients with an ECOG PS of 0-2 (P=0.036) (Online Supplementary Figure S2B),
Molecular analysis encompassed sequencing of 64 genes recur- rently mutated in AML. We analyzed either known mutational hotspots or the entire coding sequence using an amplicon-based approach (Haloplex, Agilent, Boeblingen, Germany) as described
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