Elsevier

Leukemia Research

Volume 56, May 2017, Pages 7-12
Leukemia Research

Coexisting and cooperating mutations in NPM1-mutated acute myeloid leukemia

https://doi.org/10.1016/j.leukres.2017.01.027Get rights and content

Highlights

  • NPM1 mutations in acute myeloid leukemia were accompanied by additional mutations.

  • NPM1 had the highest variant allele fraction (VAF) in only one of 120 cases.

  • Nearly all NPM1-mutated AML patients showed a pattern of concurrent gene mutations.

  • Concurrent gene mutations had higher median VAFs than the co-existing NPM1 mutation.

Abstract

NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n = 120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0–11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.

Introduction

Nucleophosmin gene (NPM1) mutations represent a relatively common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. Nucleophosmin is a multifunctional phosphoprotein that shuttles between the nucleus and the cytoplasm [1]. It plays an important role in diverse cellular processes including ribosome biogenesis, regulation of apoptosis, DNA repair, and maintenance of genome stability [2]. All pathogenic NPM1 mutations in AML occur in exon 11 (formerly identified as exon 12) and almost always result in a net insertion of four base pairs. The exact functional consequences of the mutations are unclear, but the resulting abnormal protein product is restricted to the cytoplasm and appears to act in a dominant negative fashion, inhibiting wild type NPM1 [3]. Detection of abnormal cytoplasmic accumulation of NPM1 in myeloblasts can be used as a surrogate marker of the presence of a mutation.

In AML patients, NPM1 mutations are independently associated with favorable outcome [4], [5]. However, mutated NPM1 is almost uniformly associated with other known driver mutations and patient outcome can be further refined by the overall pattern of mutations. For example, patients with mutated NPM1 along with FLT3-internal tandem duplication (FLT3-ITD) and mutated DNMT3A have a worse outcome compared to patients with any two of these abnormalities [4], [6]. Thus, for comprehensive prognostication it is necessary to examine the overall genetic context of mutated NPM1 by evaluating other known driver mutations in myeloid malignancies, typically through the use of next generation sequencing (NGS) panels [7].

NPM1 mutations, by themselves, may represent an AML-inciting genetic lesion. This assertion is supported by NPM1 mutant mouse models and the fact that they are not commonly observed with recurrent AML-associated translocations [8], [9]. AML with mutated NPM1 is a distinct entity in the 2016 World Health Organization (WHO) classification system and has been validated as a marker of minimal residual disease. In patients with a prior diagnosis of AML, the detection of low-level NPM1 mutant transcripts is associated with relapse and poor outcome [10], [11]. Thus, NPM1 mutations may perhaps be more analogous to recurrent cytogenetic abnormalities such as t(8;21), inv(16), and t(15;17) as an AML-defining genetic event, rather than alongside mutations in other genes commonly observed in AML [12]. However, this concept is not currently reflected in the WHO classification system [13].

In contrast to NPM1, mutations in genes such as ASXL1, TET2, DNMT3A and others have been observed in patients with clonal hematopoiesis of indeterminate potential (CHIP) and do not appear to be sufficient by themselves to cause disease [14]. However, they are associated with a markedly increased risk for subsequent development of a hematologic malignancy [15]. This is likely due to a competitive advantage in the clonal population and perhaps an increased susceptibility to the acquisition of additional genetic damage. Mutations in many of these genes are commonly observed to coexist with NPM1 mutations in patients with AML implying that there is a discrete series of events that occurs prior to clinically recognizable disease. The reconstruction of this sequence of events provides insight into the mechanism of leukemogenesis. Variant allele fractions (VAFs) provided by NGS-based testing can be informative in this regard. VAFs represent the fraction of specific mutant sequences (the so-called “reads” provided by NGS) relative to total sequenced reads. For example, a heterozygous autosomal variant present in all cells in a sample would have a VAF of 0.5 (50%). Using a simplified model, the original “founding” mutations can be identified based on their high VAF whereas mutations acquired later in the development of disease would have a distinctly lower VAF. Using this data, the clonal hierarchy and mutation order can be inferred and coarsely recreated. In support of this model, distinct subclones have been identified in AML samples using VAFs derived from NGS in other studies [4], [16], [17].

Several recent studies have exploited advances in massively parallel sequencing technology in an effort to shed light on the broader genetic landscape of de novo AML in adults [16], [18]. Patterns have emerged involving recurrent mutations in sets of genes which play important functional roles in basic cell biology pathways including DNA methylation and transcription, RNA splicing, cell signaling, and chromatin modification. While global data involving large cohorts of AML patients is rapidly accumulating, focused investigation of specific biologic subtypes such as NPM1-mutated AML patients is relatively lacking. In this study we sought to evaluate a large series of NPM1-mutated AML cases sequenced on an NGS-based mutation panel to assess for patterns of co-mutations associated with the subclonal architecture of NPM1-mutated AML.

Section snippets

Case identification

This study was approved by the University of Utah Institutional Review Board. Our laboratory information system was retrospectively queried for AML patients for whom we had data from a myeloid NGS panel, previously described [7]. Within this cohort, we searched for all patients with an NPM1 insertion mutation and further analyzed these cases (n = 120). No identified cases were excluded from the analysis. Cytogenetic and clinical outcome data was not available for the majority of cases and thus

Results

There were 120 NPM1-mutated AML cases identified for analysis. The median age was 62.7 years (range 21–86 years) with a female to male ratio of 1.4. NPM1-mutated cases showed an expected distribution of NPM1 mutation subtypes. The majority demonstrated the common TCTG insertion mutation designated type A (c.860_863dup, n = 84, 70%), while fewer type B (c.863_864insCATG, n = 3, 2.5%) and type D (c.863_864insCCTG, n = 2, 1.7%) mutants were observed. A variety of less common variants comprise the

Discussion

Here we describe an analysis of the mutational landscape in a cohort of NPM1-mutated adult AML patients using VAF data from NGS to gain insights into clonal relationships and pathogenesis. These data confirm findings reported by other groups – most recently by Papaemmaneuil and colleagues who described a genomic classification of AML in which most NPM1-mutated cases commonly showed additional mutations in genes such as DNMT3A, TET2, and FLT3 [4], [24]. The cohort we have described has a similar

Disclosure/Duality of interest

The authors have no disclosure or duality of interest to declare.

Acknowledgements

The authors thank the ARUP Genomics and Molecular Oncology laboratory staff for their technical contributions.

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