AbstractBackgroundB‐cell acute lymphoblastic leukemia (B‐ALL) is classified into subgroups based on known driver oncogenes and molecular lesions, including translocations and recurrent mutations. However, the current diagnostic tests do not identify subtypes or oncogenic lesions for all B‐ALL samples, creating a heterogeneous B‐ALL group of unknown subtypes.MethodsWe sorted primary adult B‐ALL cells and performed transcriptome analysis by bulk RNA sequencing (RNA‐seq).ResultsTranscriptomic analysis of an adult B‐ALL cohort allowed the classification of four patient samples with subtypes that were not previously revealed by standard gene panels. The leukemia of two patients were of the DUX4 subtype and two were CRLF2⁺ Ph‐like B‐ALL. Furthermore, single nucleotide variant analysis detected the oncogenic NRAS‐G12D, KRAS‐G12D, and KRAS‐G13D mutations in three of the patient samples, presenting targetable mutations. Additional oncogenic variants and gene fusions were uncovered, as well as multiple variants in the PDE4DIP gene across five of the patient samples.ConclusionWe demonstrate that RNA‐seq is an effective tool for precision medicine in B‐ALL by providing comprehensive molecular profiling of leukemia cells, identifying subtype and oncogenic lesions, and stratifying patients for appropriate therapy.