Integrative Genomics Approaches Identify Novel Disease-Related Genetic Aberrations in Acute Myeloid Leukemia

Abstract 402

Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of acquired somatic genetic alterations in hematopoietic progenitor cells that alter mechanisms of self-renewal, proliferation and differentiation. While recently numerous aberrations have been identified, many more mutations involved in the multistep pathogenesis of AML still remain to be discovered.

Based on critical regions and differentially expressed genes identified by our SNP/aCGH and microarray-based gene expression profiling analysis of 320 AML cases, we designed a custom capture microarray (Nimblegen, Roche) to enrich the genomic DNA of the entire coding region of 1000 genes of potential leukemia-relevance. In total, we captured and sequenced the respective genes in paired diagnosis and remission samples of 50 AML cases using Next Generation Sequencing (NGS) technology (Illumina GAIIx). We analyzed a representative AML sample cohort (n=50), which included 19 cytogenetically normal (CN) AML cases already known routinely determined mutations (CEBPA, NPM1, FLT3, and WT1), 10 CN-AML cases without known mutations, 7 complex karyotype AML cases, 13 core-binding-factor (CBF) AML cases with t(8;21) or (inv16), and one case with translocation t(4;11).

On average, we generated 9.5 million reads per sample, with 28.7% of the reads uniquely mapping to the target regions. The mean target coverage per base analyzed was 58.5-fold. For the analysis of our NGS data, we established a pipeline that allows the detection of single nucleotide variations (SNVs) and insertion/deletions (indels). A first analysis using this pipeline already provided novel insights. We detected on average 3 somatic protein altering SNVs previously not reported as SNPs and 1.3 indels per sample, which affected mostly histone-modifying enzymes, chromatin-organizing molecules or transcriptional factors, suggesting importance and at the same time variability of epigenetic changes underlying AML. We found somatic mutations in genes already known to be involved in epigenetic deregulation of leukemias (TET2, TET1), but also identified novel mutations in genes involved in the regulation of the chromatin structure (such as SATB1 or HIST1H2AA). Notably, overrepresentation analysis of the genes affected by missense SNVs revealed the enrichment of gene sets involved in the chromosome organization, DNA repair, and chromatin modification.

In conclusion, while targeted NGS identified many aberrations in AML, further analysis will identify recurrent “driver” mutations that play a pivotal role in the pathomechanism of AML and, thus, might enable better targeted therapeutic approaches, especially with regard to the guidance of epigenetic therapies.