Gene Expression Profiling In Leukemic Stem Cell-Enriched AML CD34⁺ Cell Fraction Identifies Target Genes That Predict Prognosis In Normal Karyotype AML

Abstract 952

Acute myeloid leukemia (AML) is clinically, cytogenetically and molecularly a heterogeneous disease which makes it challenging to classify it properly. In recent years major advances have been achieved in predicting outcome. However, there is still need for more powerful and independent prognostic factors that can guide treatment decisions, especially for the large subgroup of patients presenting with normal karyotype AML. In order to improve the identification of prognostic markers, gene expression studies have been performed. However, most of these studies have analyzed the mononuclear cell fraction. So, very little has been revealed about gene expression programs that drive leukemic transformation in the small population of leukemic stem cells (LSCs). Although considerable heterogeneity appears to exist in the phenotype of LSCs, CD34 is uniformly expressed and LSCs have been found to reside in the CD34⁺ compartment in the vast majority of leukemias. In the present study AML CD34⁺-specific gene expression profiles were indentified and used to distinguish prognostically relevant target genes in normal karyotype AML. AML mononuclear cells (n=46) were sorted in CD34⁺ and CD34^- subfractions and genome-wide expression analysis was performed using Illumina BeadChip Arrays. AML CD34⁺ and CD34^- gene expression was compared to a large group of normal CD34⁺ bone marrow cells (n=31). Unsupervised hierarchical clustering analysis showed that CD34⁺ AML samples belonged to a distinct cluster compared to normal bone marrow and that in 61% of the cases the AML CD34⁺ transcriptome did not cluster together with the paired CD34^- transcriptome. These data indicate that in the majority of AML cases the leukemic stem cell-enriched CD34⁺ gene expression profile is quite distinct from the leukemic CD34^- compartment. GO analysis revealed that common differences in gene expression between CD34⁺ and CD34^- groups were particularly enriched for genes that were associated with T-cells and erythropoiesis. This association with a more committed phenotype was found in all AML samples and not just in those samples where CD34⁺ and CD34^- transcriptomes did not cluster together. Among the GO-ontologies representing the differentially expressed genes between CD34⁺ AML versus CD34⁺ normal bone marrow cells were gene sets related to DNA damage and a number of mitotic and metabolic processes. A top 50 of AML CD34⁺-specific genes was selected by comparing the AML CD34⁺ transcriptome with the AML CD34^- and CD34⁺ normal bone marrow transcriptomes. The prognostic relevance of these 50 genes was assessed using univariate cox regression analyses between the continuous transcript levels of these 50 genes and overall survival (OS) in a large series of normal karyotype AML (n=163) (Metzeler et al. Blood 2008). The findings were validated in another independent cohort of 218 normal karyotype AML patients (Valk et al. NEJM 2004). Interestingly, higher transcript levels of three CD34⁺ AML specific genes, i.e. ankyrin repeat domain 28 (ANKRD28), guanine nucleotide binding protein, alpha 15 (GNA15) and UDP-glucose pyrophosphorylase 2 (UGP2) were associated with a significant poorer OS in both cohorts (p<0.01). For the cohort of 218 normal karyotype AML patients also event free survival (EFS) data were available for further analyses. A significant association between the continuous transcript levels of ANKRD28, GNA15 and UGP2 with poor EFS was evident. Also for the sum of expression of ANKRD28, GNA15 and UGP2 higher transcript levels were strongly associated with poorer OS (p=0.007) and EFS (p=0.006) in the cohort of 218 normal karyotype AML. Similar results were obtained in the cohort of 163 normal karyotype AML patients for OS (p<0.001). Importantly, the prognostic value of the continuous transcript levels of these three genes was independent from the well known risk factors FLT3-ITD, NPM1c⁺ and CEBPA mutation status as determined by a multivariate analysis in the cohort of 218 normal karyotype AML patients. In conclusion, by microarray analysis of the leukemic stem cell-enriched AML CD34⁺ cell fraction novel insight was obtained in gene expression programs that are potentially associated with leukemic stem cell self-renewal and transformation. Moreover, the identified new gene expression profiles were shown to have prognostic relevance in normal karyotype AML independent of well known risk factors.