Analysis of rare driving events in pediatric acute myeloid leukemia

Abstract

Elucidating genetic aberrations in pediatric acute myeloid leukemia (AML) provides insight in biology and may impact on risk-group stratification and clinical outcome. This study aimed to detect such aberrations in a selected series of samples without known (cyto)genetic aberration using molecular profiling. A cohort of 161 patients was selected from various study groups: DCOG, BFM, SJCRH, NOPHO and AEIOP. Samples were analyzed using RNA sequencing (n=152), whole exome (n=135) and/or whole genome sequencing (n=100). In 70 of 156 patients (45%), of whom RNA sequencing or whole genome sequencing was available, rearrangements were detected, 22 of which were novel; five involving ERG rearrangements and four NPM1 rearrangements. ERG rearrangements showed self-renewal capacity in vitro, and a distinct gene expression pattern. Gene set enrichment analysis of this cluster showed upregulation of gene sets derived from Ewing sarcoma, which was confirmed comparing gene expression profiles of AML and Ewing sarcoma. Furthermore, NPM1-rearranged cases showed cytoplasmic NPM1 localization and revealed HOXA/B gene overexpression, as described for NPM1 mutated cases. Single-gene mutations as identified in adult AML were rare. Patients had a median of 24 coding mutations (range, 7-159). Novel recurrent mutations were detected in UBTF (n=10), a regulator of RNA transcription. In 75% of patients an aberration with a prognostic impact could be detected. Therefore, we suggest these techniques need to become standard of care in diagnostics.

Figure 1.

Flow diagram of the cohort selection. Patients from different cohorts were selected according to the research groups diagnostic procedures. Patient samples from SJCRH were selected based on karyotype analysis only, patient samples from DCOG and BFM were selected based on karyotype analysis and molecular analysis such as reverse transcription polymerase chain reaction (RT-PCR) and KMT2A split-fluorescence in situ hybridization analysis. NOPHO and AIEOP both provided samples from patients with a normal karyotype, of whom NOPHO excluded NPM1 mutated patients. In total 161 patients had good quality RNA sequencing (RNAseq) (n=152), whole exome sequencing (WES) (n=135) and/or whole genome sequencing (WGS) (n=100). SJCRH: St Jude Children’s Research Hospital; DCOG: Dutch Childhood Oncology Group; BFM: Berlin-Frankfurt-Münster Group; NOPHO: Nordic Society of Pediatric Hematology and Oncology; AIEOP: Associazione Italia di Ematologica e Oncologia Pediatrica. FISH: Fluorescence in situ hybridization; mut: mutated; rearr: rearrangement.

Figure 2.

Integrative analysis of mutations and gene expression. (A) Waterfall plot of recurrent mutations occurring in at least 4 patients, mutations occurring in less than 3 patients were excluded from this analysis. The plot was first ordered based on type II aberration. Rearrangements occurring less than 5-times were taken together. From FLT3 downward, genes were ordered based on the number of mutations that occurred. (B) t-distributed stochastic neighbor embedding (T-SNE )analysis using the top 200 most variant genes based on standard deviation of 153 patients with RNA-sequencing data. ITD: internal tandem repeat; rearr: rearrangement.

Figure 3.

Patient outcome. (A) Event-free survival (EFS) and (B) overall survival (OS) of patients stratified by driving aberration. Aberrations that occur less than 5-times in this cohort have been combined with other rearrangements.

Figure 4.

Characteristics of ERG-rearranged acute myeloid leukemia. (A) Survival curves combining data from Bolouri et al. and this cohort, comparing survival of ERG-rearranged acute myeloid leukemia (AML) to the remaining cohort. (B) Gene set enrichment analysis plots of gene sets derived from EWSR1-FLI1 targets and embryonic stem cells. (C) Hierarchical clustering of AML and Ewing sarcoma samples using the top 50 differentially expressed genes of ERG-rearranged AML vs. other AML (absolute log-fold change). Ewing sarcoma samples cluster together with ERG-rearranged AML. (D) Colony-forming assay of ERG and FEV rearrangments detected in AML. NUP98-KDM5A was taken along as a positive control. Expression of EWSR1-ERG and FUS-ERG in murine lin- cells resulted in repetitive replating. EFS: event-free survival; OS: overall survival.

Figure 5.

Characteristics of NPM1-rearranged acute myeloid leukemia. (A to C) Gene set enrichment analysis plots of gene sets upregulated in samples with an event in NPM1 compared to samples without an event in NPM1 (NPM1 wild-type [wt]), of which (A) depicts all NPM1 events (both rearrangements and mutations) vs. NPM1 wt, (B) depicts NPM1 mutations vs. NPM1 wt, and (C) depicts NPM1 rearrangements (NPM1r) vs. NPM1 wt. (D). Cytospins of primary patient material stained with anti-NPM1. NPM1 is located in the nucleoli in healthy peripheral blood and in an acute myeloid leukemia (AML) sample with NPM1 wt, whereas it is localized in the cytoplasm in the NPM1-mutated and NPM1-rearranged AML samples.