Distinct patterns of mutations occurring in de novo AML versus AML arising in the setting of severe congenital neutropenia

Abstract

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Like most other bone marrow failure syndromes, it is associated with a marked propensity to transform into a myelodysplastic syndrome (MDS) or acute leukemia, with a cumulative rate of transformation to MDS/leukemia that exceeds 20%. The genetic (and/or epigenetic) changes that contribute to malignant transformation in SCN are largely unknown. In this study, we performed mutational profiling of 14 genes previously implicated in leukemogenesis using 14 MDS/leukemia samples from patients with SCN. We used high-throughput exon-based resequencing of whole-genome-amplified genomic DNA with a semiautomated method to detect mutations. The sensitivity and specificity of the sequencing pipeline was validated by determining the frequency of mutations in these 14 genes using 188 de novo AML samples. As expected, mutations of tyrosine kinase genes (FLT3, KIT, and JAK2) were common in de novo AML, with a cumulative frequency of 30%. In contrast, no mutations in these genes were detected in the SCN samples; instead, mutations of CSF3R, encoding the G-CSF receptor, were common. These data support the hypothesis that mutations of CSF3R may provide the "activated tyrosine kinase signal" that is thought to be important for leukemogenesis.

Figure 1

NRAS exon 2 sequence coverage maps. Sequence coverage for exon 2 of NRAS is shown for the Discovery set of 94 AML samples (both tumor and skin), the 94 CALGB AML samples, and the 22 SCN-AML samples; each row represents an individual sample. Each column represents a single nucleotide position starting from the 5′-intronic region (■), through exon 2 of NRAS (□) and 3′-intronic region (■). The position of codons 12 and 13 of NRAS is highlighted. High-quality double-stranded sequence is shown in green, single-stranded coverage in yellow, and no coverage in red.

Figure 2

Nucleotide changes for the discovery set of de novo AML samples. Nonsynonymous nucleotide changes and gene deletions or insertions are highlighted in red; white boxes indicate missing sequence data. Known SNPs are excluded from this analysis. Both leukemia (T) and germline (G) samples for each patient were sequenced. ITD indicates internal tandem duplication (FLT3); INS, tetranucleotide insertion of exon 12 (NPM1).

Figure 3

Nucleotide changes in the SCN samples. Nonsynonymous nucleotide changes and gene deletions or insertions are highlighted in red; white boxes indicate missing sequence data. Known SNPs are excluded from this analysis. Pre- and post-leukemic samples from the same patient are outlined.