Analysis of Loss of Heterozygosity in Adult Patients with De Novo Acute Myeloid Leukemia and Normal Karyotype.

Abstract

A large proportion of acute myeloid leukemia (AML) exhibits a normal karyotype in which the underlying pathomechanisms still have to be determined. Novel techniques like arrayCGH or single nucleotide polymorphism (SNP) chip analysis allow the identification and characterization of molecular rearrangements at the sub-megabase level. Recently, the application of genome-wide SNP array technology revealed frequent uniparental disomy (UPD) in approximately 20% of AML suggesting that UPD represents a nonrandom event in leukemogenesis. Uniparental disomy is acquired by somatic recombination and therefore not accessible by conventional cytogenetic methods or arrayCGH. In this study we analyzed DNA from AML patients with normal karyotype for the presence of LOH. SNP analysis was performed on the Mapping 100k GeneChip (Affymetrix, Santa Clara, CA). DNA was extracted from paired samples of 56 de novo AML patients with normal karyotype at diagnosis and in complete remission, respectively. Signal intensity data were analyzed by the GCOS GeneChip analysis software and statistical analysis of SNP call data was performed by the dChipSNP software. In addition, standard mutation screening of the genes encoding NPM1, FLT3, CEBPA, MLL and NRAS was performed in all cases. Using the 100k SNP array, a mean SNP call rate of 98.2% was reached, resulting in > 110,000 SNP genotype calls per sample. Signal intensity data analysis revealed submicroscopic chromosomal deletions resulting in hemizygosity in three patients. Patient 1 had a single 2 Mb deletion in chromosomal band 3p14.1, patient 2 had two small deletions affecting chromosome 12q23 and 12p13, the latter encompassing the ETV6 locus, and patient 3 had two small deletions within the long arm of chromosome 8. Besides these small chromosomal regions of copy number alterations, we found 4 large stretches of somatically acquired homozygosity without numeric alterations, affecting chromosome 6 (6p21 to 6 pter and 6q26 to 6 qter), chromosome 11 (11p12 to 11pter) and chromosome 13 (13q11 to 13qter). Noteworthy, in the case with uniparental disomy of chromosome 13, we could detect a homozygous FLT3-ITD mutation, supporting the findings that acquired isodisomy for chromosome 13 is common in AML, and associated with FLT3-ITD mutations (Griffiths et al., Leukemia, 2005). In summary, high resolution SNP assay technology in AML patients with normal karyotype allowed the identification of distinct chromosomal regions affected by UPD, supporting the postulated nonrandom mechanism of acquired mitotic recombination events in AML. Besides known chromosomal regions known to be affected by genomic aberrations in AML, we found additional submicroscopic chromosomal aberrations in cases with normal karyotype. Analysis of larger patient series will allow the identification of novel regions of interest harboring genes that might be involved in the pathogenesis of AML.