RNA-based next-generation sequencing for identification of clinically actionable gene fusions missed by conventional cytogenetics in hematologic malignancies.

e18534 Background: Accurate detection of genomic alterations with diagnostic, prognostic, and therapeutic relevance is critical in hematologic malignancies. Conventional cytogenetic methods, including karyotyping and fluorescence in situ hybridization (FISH), are routinely used but may fail to detect cryptic or complex gene fusions. European LeukemiaNet (ELN) guidelines recommend assessment for KMT2A rearrangements due to their prognostic significance. RNA-based next-generation sequencing (RNA-NGS) interrogates expressed fusion transcripts and may identify clinically actionable alterations missed by standard cytogenetic testing. We evaluated the clinical utility of RNA-NGS in patients with negative conventional cytogenetic results. Methods: We retrospectively reviewed RNA-NGS testing performed in patients with hematologic malignancies at our institution between 2022 and 2024. Among 194 patients with positive RNA-NGS results, we identified cases with clinically relevant alterations detected despite negative karyotype and/or FISH studies. Analyses were conducted at the patient level to assess potential impact on risk stratification and treatment decisions. Results: RNA-NGS identified clinically relevant genomic alterations in a substantial subset of patients with negative conventional cytogenetic testing. At the patient level, 23% harbored actionable RNA-NGS findings despite both negative karyotype and FISH results, with additional patients demonstrating RNA-NGS–positive findings in the setting of either negative karyotype or negative FISH alone. Discordant cases most commonly involved cryptic or complex gene fusions with established clinical significance, including ELN-relevant rearrangements such as KMT2A::ELL and KMT2A::MLLT4 , as well as NUP98::NSD1 , KAT6A::CREBBP , RUNX1::USP42 , and ETV6::ABL1 . Identification of these alterations would have directly informed ELN risk classification and therapeutic planning. Conclusions: RNA-NGS detects clinically actionable gene fusions, including ELN guideline–defined KMT2A rearrangements, that are frequently missed by conventional cytogenetic methods. Notably, nearly one quarter of patients with negative karyotype and FISH results harbored significant RNA-NGS findings, highlighting a clinically meaningful false-negative rate with standard testing alone. Incorporation of RNA-NGS into routine diagnostic workflows may improve guideline-aligned risk stratification and support more informed treatment decisions in hematologic malignancies.