ORIGINAL ARTICLE Gene expression profiling in the leukemic stem cell-enriched CD34 þ fraction identifies target genes that predict prognosis in normal karyotype AML HJM de Jonge 1,7 , CM Woolthuis 2,7 , AZ Vos 2 , A Mulder 3 , E van den Berg 4 , PM Kluin 5 , K van der Weide 2 , ESJM de Bont 6 , G Huls 2 , E Vellenga 2,8 and JJ Schuringa 2,8 1 Department of Experimental Hematology and Department of Pediatrics, Division of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2 Department of Experimental Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 3 Department of Clinical Chemistry, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 4 Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 5 Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands and 6 Department of Pediatrics, Division of Pediatric Oncology/Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands In order to identify acute myeloid leukemia (AML) CD34 þ - specific gene expression profiles, mononuclear cells from AML patients (n ¼ 46) were sorted into CD34 þ and CD34 À subfrac- tions, and genome-wide expression analysis was performed using Illumina BeadChip Arrays. AML CD34 þ and CD34 À gene expression was compared with a large group of normal CD34 þ bone marrow (BM) cells (n ¼ 31). Unsupervised hierarchical clustering analysis showed that CD34 þ AML samples belonged to a distinct cluster compared with normal BM and that in 61% of the cases the AML CD34 þ transcriptome did not cluster together with the paired CD34 À transcriptome. The top 50 of AML CD34 þ -specific genes was selected by comparing the AML CD34 þ transcriptome with the AML CD34 À and CD34 þ normal BM transcriptomes. Interestingly, for three of these genes, that is, ankyrin repeat domain 28 (ANKRD28), guanine nucleotide binding protein, alpha 15 (GNA15) and UDP-glucose pyrophosphorylase 2 (UGP2), a high transcript level was associated with a significant poorer overall survival (OS) in two independent cohorts (n ¼ 163 and n ¼ 218) of normal karyotype AML. Importantly, the prognostic value of the continuous transcript levels of ANKRD28 (OS hazard ratio (HR): 1.32, P ¼ 0.008), GNA15 (OS HR: 1.22, P ¼ 0.033) and UGP2 (OS HR: 1.86, P ¼ 0.009) was shown to be independent from the well-known risk factors FLT3-ITD, NPM1c þ and CEBPA muta- tion status. Leukemia (2011) 25, 1825–1833; doi:10.1038/leu.2011.172; published online 15 July 2011 Keywords: Acute myeloid leukemia; gene expression profiling; leukemic stem cells; prognostic factors; CD34 þ cells; transcriptome analysis Introduction Acute myeloid leukemia (AML) is clinically, cytogenetically and molecularly a heterogeneous disease, which makes it challenging to classify it properly. Currently, patients diagnosed with AML are stratified into separate risk groups based on morphological, cytogenetic and molecular abnormalities. 1,2 However, especially in the intermediate risk group that represents the largest AML subgroup (60%), treatment outcome varies considerably. Within the intermediate risk group, subgroups are recognized based on mutations in the nucleophosmin 1 (NPM1) (leading to cytoplas- mic dislocalization of NPM1 (NPM1c þ )) and fms-related tyrosine kinase 3 (FLT3) genes or based on biallelic mutations in the CEBP a gene. 3–5 Identification of novel molecular markers might therefore be helpful for further risk stratification. In recent years, a number of gene expression profiling (GEP) studies has been performed in order to improve the identification of known cytogenetic subgroups and to recognize new clusters of AML patients with distinct gene-expression signatures. 6–11 Most of these AML GEP studies have been performed using the total AML–mononuclear cell (MNC) fraction. 6–9 As cell lineage and differentiation stages affect gene expression-based clustering, 6,7,10 the differentially expressed genes associated with the differentia- tion stage might obscure more basic gene expression information related to tumor initiation and maintenance. Consequently, profiling of a more homogenous leukemic cell population, instead of the total MNC fraction, might enhance the feasibility of using GEP to identify novel prognostic markers. AML is thought to be initiated and maintained by relatively small numbers of leukemia-initiating cells that have an enhanced self-renewal capacity and can engraft in immuno- deficient mice. 12,13 In the vast majority of leukemias, leukemia- initiating cells have been found to reside in the CD34 þ compartment. 12–17 Studies aimed at further enrichment of leukemia-initiating cells revealed substantial heterogeneity in cell surface marker expression, 18–23 and it has been observed that leukemia-initiating cells can reside both in the CD34 þ / CD38 À and in the CD34 þ /CD38 þ fractions. 24 In our current study, AML–MNC fractions were sorted in CD34 þ and CD34 À subfractions and gene expression was compared with a large group of normal CD34 þ bone marrow (BM) cells. Thus, we were able to identify AML CD34 þ -specific gene expres- sion profiles, which included a number of genes that could significantly predict prognosis in normal-karyotype AML independent of already established prognostic factors. Materials and methods Patient material GEP was performed on blast cells of 46 patients with AML from a single center (University Medical Center Groningen). Received 15 December 2010; revised 12 May 2011; accepted 8 June 2011; published online 15 July 2011 Correspondence: Dr JJ Schuringa or E Vellenga, Department of Experimental Hematology, University Medical Center Groningen, Hanzeplein 1, Groningen, Groningen 9713 GZ, The Netherlands. E-mail: j.schuringa@int.umcg.nl or e.vellenga@int.umcg.nl 7 Shared first authorship. 8 Shared senior authorship. Leukemia (2011) 25, 1825–1833 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu