Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis.

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

Mutations in nucleophosmin NPM1 are the most frequent acquired molecular abnormalities in acute myeloid leukemia (AML). We determined the NPM1 mutation status in a clinically and molecularly well-characterized patient cohort of 275 patients with newly diagnosed AML by denaturing high-performance liquid chromatography (dHPLC

© F e r r a t a S t o r t i F o u n d a t i o nR.G.W. Verhaak et al. between both AML sample populations. Mutational analyses to detect recurrent mutations in AML were performed as previously described. [13][14][15][16] All supervised class prediction analyses were performed with Prediction Analysis for Microarrays (PAM) software version 1.28 in R version 2.1.0. 17 Clinical, cytogenetic and molecular information as well as the gene expression profiles of all primary AML cases is available at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo, accession number GSE6891). Results and DiscussionIn this study of 461 clinically and molecularly well-characterized cases of AML (Table 1), we were able to comprehensively validate the application of GEP to predict therapeutically relevant molecular subtypes in AML.We applied PAM to investigate whether karyotypic and mutational abnormalities with prognostic or therapeutic value in AML were accurately predictable based on GEP. PAM allows the selection of the minimal number of genes required for optimal prediction, which may be beneficial in a diagnostic setting. The AML cohort1 (n=247) was used as training set to derive predictive signatures that were subsequently validated on AML cohort2 (n=214). The deduced expression signatures are available in the Online Supplementary Tables S1-18.The cytogenetic status of all AML patients with favorable risk, i.e. those with t(8;21), t(15;17) or inv(16) abnormalities, was predicted with 100 percent accuracy ( Table 2). In fact, among these predicted AML cases, there were cases with favorable cytogenetics that had previously been missed by routine cytogenetics (4 out of 37 inv(16) and 4 out of 25 t(15;17)). The presence of the translocation-related fusion transcripts in these specific cases was confirmed by real-time quantitative PCR. Thus, GEP is a reliable alternative to discriminate these three AML subtypes, 2,3 which represent approximately 20% of all cases.2,3 Prediction of t(15;17) and inv(16) required only few genes, as seen previously. 8 For the t(8;21) cases, 76 probe sets were needed to correctly classify all samples. However, as few as two probe sets, including one associated with the RUNX1T1 (ETO) gene, were sufficient to accurately classify all but one t(8;21) cases, which is also consistent with earlier studies 8 (Online Supplementary Figure S3). AML cases with mutations in the transcription factor CCAAT/enhancer binding protein α (CEBPA), which are associated with a relatively favorable treatment outcome, were predicted with positive and negative predictive values of 100% and 97% respectively. Six out of 15 CEBPA mutant cases were missed in the validation set (sensitivity 60%; Table 2). Of note, the misclassified cases all carried a single heterozygous CEBPA mutation, whereas samples with biallelic mutations (either homo-or heterozygous) were all correctly recognized (data not shown). In the training cohort, all but two (14/16) samples carried biallelic mutations 14,18 and in cross-validation in the training cohort ...

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