Next-generation sequencing of acute myeloid leukemia identifies the significance of TP53, U2AF1, ASXL1, and TET2 mutations

Abstract

We assessed the frequency and clinicopathologic significance of 19 genes currently identified as significantly mutated in myeloid neoplasms, RUNX1, ASXL1, TET2, CEBPA, IDH1, IDH2, DNMT3A, FLT3, NPM1, TP53, NRAS, EZH2, CBL, U2AF1, SF3B1, SRSF2, JAK2, CSF3R, and SETBP1, across 93 cases of acute myeloid leukemia (AML) using capture target enrichment and next-generation sequencing. Of these cases, 79% showed at least one nonsynonymous mutation, and cases of AML with recurrent genetic abnormalities showed a lower frequency of mutations versus AML with myelodysplasia-related changes (P<0.001). Mutational analysis further demonstrated that TP53 mutations are associated with complex karyotype AML, whereas ASXL1 and U2AF1 mutations are associated with AML with myelodysplasia-related changes. Furthermore, U2AF1 mutations were specifically associated with trilineage morphologic dysplasia. Univariate analysis demonstrated that U2AF1 and TP53 mutations are associated with absence of clinical remission, poor overall survival (OS), and poor disease-free survival (DFS; P<0.0001), whereas TET2 and ASXL1 mutations are associated with poor OS (P<0.03). In multivariate analysis, U2AF1 and TP53 mutations retained independent prognostic significance in OS and DFS, respectively. Our results demonstrate unique relationships between mutations in AML, clinicopathologic prognosis, subtype categorization, and morphologic dysplasia.

Figure 1

Mutational patterns in acute myeloid leukemia (AML). (a) Unsupervised gene cluster analysis of 19 genes (RUNX1, ASXL1, TET2, CEBPA, IDH1, IDH2, DNMT3A, FLT3, NPM1, TP53, NRAS, EZH2, CBL, U2AF1, SF3B1, SRSF2, JAK2, CSF3R, and SETBP1) in association with AML subtypes. (b) Overall mutational burden and frequency in AML with myelodysplasia-related changes (AML-MRC), AML-not otherwise specified (AML-NOS), AML with recurrent genetic abnormalities (AML-RGA), and therapy-related AML (AML-T). Comparison demonstrates that AML-MRC shows increased frequency of mutations and higher number of genes mutated, whereas AML-RGA shows fewer mutations. (c–e) The mutational distribution of 19 genes in AML subtypes; depicted are the frequency in each subtype.

Figure 2

Frequency of mutations in acute myeloid leukemia (AML) by cytogenetic risk stratification. (a) AML with low-risk cytogenetics, (b) AML with intermediate-risk cytogenetics, and (c) AML with high-risk cytogenetics.

Figure 3

TP53 mutations are associated with complex karyotype, whereas U2AF1 mutations are associated with acute myeloid leukemia (AML) with myelodysplasia-related changes (MRC) and trilineage dysplasia. (a) Cases of AML with complex karyotype vs cases without complex karyotype. Black bars indicate cases with TP53 mutated, whereas gray bars indicate cases without TP53 mutated. Complex karyotype cases include an additional 21 cases studied for TP53 mutations by Sanger sequencing. (b) Cases of AML by WHO subtype including AML-MRC, AML-not otherwise specified (AML-NOS), AML with recurrent genetic abnormalities (AML-RGA), and therapy-related AML (AML-T). The black bar indicates cases with U2AF1 mutated, and gray bars indicate cases without U2AF1 mutated. (c) An example of an AML with U2AF1 mutated that shows profound trilineage dysplasia including megakaryocytes with separate nuclear lobes (black arrows), hypogranular neutrophils (black arrowhead), and erythroids with nuclear membrane irregularities (red arrow). Blasts are identified by red arrowheads.

Figure 4

Kaplan–Meier curves for TP53, U2AF1, and TET2. (a) Overall survival (OS) Kaplan–Meier curve for TP53. (b) OS Kaplan–Meier curve for U2AF1. (c) OS Kaplan–Meier curve for TET2. (d) OS Kaplan–Meier curve for ASXL1. (e) Disease-free survival (DFS) Kaplan–Meier curve for TP53. (f) DFS Kaplan–Meier curve for U2AF1. (g) DFS Kaplan–Meier curve for TET2. (h) DFS Kaplan–Meier curve for ASXL1.

Figure 5

Prognostic significance of TP53 mutations in cases of acute myeloid leukemia (AML) with complex karyotype. (a) Kaplan–Meier curve of disease-free survival (DSF) in AML with complex karyotype with relation to TP53 mutations. (b) Kaplan–Meier curve of overall survival (OS) in AML with complex karyotype with relation to TP53 mutations. Cases included those sequenced by next-generation sequencing and TP53 Sanger sequencing.