Inappropriate expression of EVI1 (ecotropic virus integration-1), in particular splice form EVI1-1D, through chromosome 3q26 lesions or other mechanisms has been implicated in the development of high-risk acute myeloid leukemia (AML). To validate the clinical relevance of EVI1-1D, as well as of the other EVI1 splice forms and the related MDS1/EVI1 (ME) gene, real-time quantitative polymerase chain reaction was performed in 534 untreated adults with de novo AML. EVI1-1D was highly expressed in 6% of cases (n ؍ 32), whereas 7.8% were EVI1 ؉ (n ؍ 41) when all splice variants were taken into account. High EVI1 predicted a distinctly worse event-free survival (HR ؍ 1.9; P ؍ .002) and disease-free survival (HR ؍ 2.1, P ؍ .006) following multivariate analysis. Importantly, we distinguished a subset of EVI1 ؉ cases that lacked expression of ME (EVI1 ϩ ME Ϫ ; n ؍ 17) from cases that were ME ؉ (EVI1 ϩ ME ϩ ; n ؍ 24). The atypical EVI1 ϩ ME Ϫ expression pattern exhibited cytogenetically detectable chromosomal 3q26 breakpoints in 8 cases. Fluorescence in situ hybridization revealed 7 more EVI1 ϩ ME Ϫ cases that carried cryptic 3q26 breakpoints, which were not found in the EVI1 ϩ ME ϩ group. EVI1 ϩ ME Ϫ expression predicts an extremely poor prognosis distinguishable from the general EVI1 ϩ AML patients (overall survival