Targeted Next-Generation Sequencing (NGS) Enables a Broad Screening of 95 Molecular Mutations In a One-Step Approach and Detects Recurrent Mutations In AML with a Normal Karyotype In MYH11 and NOTCH1

Abstract

AbstractAbstract 175Acute myeloid leukemia (AML) is a heterogeneous disease and based on cytogenetic stratification AML with a normal karyotype (AML-NK) is an intermediate-risk group. In recent years the detection of novel molecular mutations allowed to further stratify AML-NK into different prognostic subgroups. However, thus far still in a subset of AML-NK no recurrent mutations have been identified. In order to detect novel recurrent mutations we performed DNA sequence enrichment from complex genomic samples using microarrays to enable a targeted Next-Generation Sequencing (NGS) analysis. We combined 454 PicoTiterPlate (PTP) pyrosequencing with long-oligonucleotide sequence capture arrays to apply this technique for the comprehensive molecular genetic characterization of AML-NK. 6 bone marrow specimens from untreated de novo AML patients at diagnosis were analyzed (n=4 AML-NOS; n=2 AML-MRC according to WHO classification). All cases were shown to be negative for the most frequent mutations in FLT3 (both internal tandem duplication and tyrosine kinase domain), MLL partial tandem duplication, NPM1, and CEBPA. A custom 1.91 Mb microarray was designed to contain capture probes for all coding regions of 95 genes, in total 1608 exons (NimbleGen 385K format; Madison, WI). These 95 target genes had been selected according to their relevance in leukemogenic pathways, i.e. cell cycle control, cell proliferation and differentiation, multidrug resistance, growth factor receptors, oncogenes, tumor suppressors, and homeobox genes. Starting with 20 μ g of genomic DNA, this array design allowed a median 294-fold DNA enrichment of these targeted genomic loci, as assessed by ligation-mediated LM-PCR. Three patients per lane were sequenced on a 2-lane PTP using the large volume Titanium chemistry assay (454 Life Sciences, Branford, CT). Each case was tagged by a molecular 10-base barcode. Overall, 1,070,724 reads with a median length of 352 bp were generated in the two lanes (GS Reference Mapper software version 2.3). In median, 72.3% on-target bases were derived from the capture array probes, resulting in a 16-fold median coverage. In total, in this proof-of-principle cohort of 6 patients, in median 1534 variants per case were detected. After excluding single nucleotide polymorphisms and noncoding aberrations, 13 nonsynonymous mutations were found in 11/95 genes analyzed (1-4 mutations per case). Single missense mutations were found for transcriptional repressor SPEN (Q340R), histone acetyltransferase and transcriptional coactivator CREBBP (K622R), multi-drug resistance transporter ABCC1 (R633Q), epidermal growth factor EGF (A995P), runt-related transcription factor RUNX1 (G138S) and mixed-lineage leukemia gene MLL (D2890G). A TET2 nonsense mutation was observed in exon 5 (E1178X), being located in a conserved domain as described by Delhommeau et al. (N Engl J Med. 2009 360:2289-301). In addition, in two patients two genes were recurrently hit by a mutation: MYH11 and NOTCH1. MYH11 missense mutations were detected in exons 36 and 39 (E1840D; M1661V). We further observed two missense NOTCH1 mutations in exons 2 and 13 (P22S; E694K), located in the extracellular epidermal growth factor-like repeats domain which is required for ligand interaction. Thus far, activating NOTCH1 mutations have been reported in the context of T cell acute lymphoblastic leukemia (heterodimerization and PEST domains). In conclusion, we demonstrated that the combination of a targeted DNA sequence enrichment assay followed by NGS technology enabled a molecular characterization of 95 genes in AML-NK in a one-step approach. New recurrent aberrations such as NOTCH1 mutations are interesting targets for a broader screening in AML subtypes. In particular, methods like this will enable an unbiased comprehensive genetic characterization of leukemias and other malignancies and are suitable to identify markers to further stratify AML-NK into different risk groups. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Roche Diagnostics GmbH: Research Funding.