Targeted sequencing

The sequencing panel targets a ~250-kb genomic region, which comprises the entire coding sequences of 126 genes that are recurrently mutated in acute leukemia.8 Mononuclear cells were enriched from pretreatment bone marrow by Ficoll density gradient centrifugation. Nimble Design GenSeq Cap EZ Choice was performed in accordance with the manufacturer’s protocol. With an Illumina HiSeq 2500, multiplexed libraries were sequenced using 100-bp paired-end runs. Reads were aligned to human genomic reference sequences using the Burrows-Wheeler alignment tool (HG19, NCBI built 37). To identify single nucleotide polymorphisms and short insertions and deletion, MuTect2 was performed with recommended parameters. A subset of somatic mutations was selected randomly for validation using Sanger sequencing.

Efficacy evaluation

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Routine blood cell counts were performed twice each week after chemotherapy. Three to four weeks after chemotherapy, bone marrow aspiration was performed and the responses to treatment were evaluated.

OS was measured from the time of diagnosis to death from any cause. EFS was measured from the time of first complete response to leukemia relapse. For secondary endpoints, bone marrow biopsies and aspirates were obtained from patients at the time of screening.

The nature of response was defined in accordance with the criteria of the International Working Group.14 Specifically, for a complete response the patient demonstrated <5% bone marrow myeloblasts, no myeloblasts with Auer rods, the absence of extramedullary disease, an absolute neutrophil count >1×109/L, and a platelet count ≥100×109/L. A complete response with incomplete blood count recovery was diagnosed when the patient had <5% bone marrow myeloblasts, no myeloblasts with Auer rods, the absence of extramedullary disease, but with incomplete blood cell recovery. A partial response was defined as a decrease of ≥50% (ie, to 5–25% total) in the myeloblasts detected in bone marrow aspirates, and those with normalized blood counts. No response was the absence of both complete and partial response. Relapse was the reappearance of leukemia cells in the peripheral blood, or >5% myeloblasts in the bone marrow. Induction death was defined as death occurring before response evaluation, unless evidence of resistant disease was provided at least 7 days after the conclusion of the chemotherapy.

Neutrophil and platelet recovery were defined, respectively, as absolute neutrophil count >0.5×109/L and platelet count >30×109/L, for 3 consecutive days. The time to neutrophil or platelet recovery was the time to the first day of 3 consecutive days of recovery. Toxicities were assessed in accordance with the National Cancer Institute Common Toxicity Criterion Version 3.14

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Statistical analyses

We studied associations between various gene mutations and patient clinical characteristics, using Fisher’s exact test or chi-squared tests for categorical endpoints (eg, response), and the Wilcoxon rank-sum test for continuous variables. Analyses of treatment outcomes were based on commonly accepted definitions of complete remission, OS, and EFS. P-values were calculated using the Kaplan–Meier method for survival analyses. A Cox proportional hazard model was used to assess the prognostic significance of the genetic mutations and clinical variables. To investigate clinical features and genetic mutations predicting outcomes after DCAG or IA induction chemotherapy, logistic and Cox multivariable analyses of the entire cohort for EFS and OS were performed, including treatment arm as a covariate. All analyses were performed with GraphPad Prism 5 software. Statistical analyses were conducted with SPSS 19.0. P<0.05 was considered statistically significant.

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