Gene Conversion Is a Late Event in Evolution of High Risk AML.

Evolution and progression of AML is considered a result of cooperation of multiple genetic and epigenetic alterations. FLT3 internal tandem duplication (ITD) is a common mutation in AML and is considered a secondary, proliferative event in the pathogenesis of AML. We used SNP/CGH microarrays to evaluate the mechanism of disease evolution in FLT3/ITD-positive AML. Diagnostic DNA from patients with FLT3/ITD (N=25), FLT3 activation loop mutation (FLT3/ALM, N=15) or with no FLT3 mutations (FLT3/WT, N=55) was evaluated for copy number and LOH alterations. 12/25 (48%) FLT3/ITD-positive patients had loss of heterozygosity (LOH) of a large segment of 13q arm downstream of the FLT3 gene without an associated chromosome loss, demonstrating a copy neutral LOH (CN-LOH) state. In contrast, CN-LOH was not detected in those with FLT3/ALM or FLT3/WT suggesting that FLT3/ITD is causally associated with evolution of CN-LOH. Single cell analysis of the FACS purified leukemic blasts from FLT3/ITD-positive samples that were heterozygous for 13q (no CN-LOH) by SNP/CGH arrays demonstrated the presence of 3 distinct molecular subtypes that included a subpopulation with CN-LOH, as well as heterozygous ITD and FLT3/WT, demonstrating oligoclonal nature of the leukemic blast population which prevented identification of CN-LOH. In patient samples with CN-LOH, CFU-GM and BFU-E colonies derived from CD34+/CD33- progenitors demonstrated heterozygous ITD, with no LOH or copy number alterations. In contrast, more differentiated, CD33+ leukemic blasts as well as CFU-GM colonies derived from CD34+/CD33+ progenitors had homozygous ITD and CN-LOH. This cumulative data demonstrates that CN-LOH of the 13q, as a result of gene conversion-type event mediates the pathogenesis of FLT3/ITD and is a secondary event in the evolution of high risk FLT3/ITD. Gene conversion, leading to haplo-insufficiency may be an important event in the evolution of AML.