Detection of BCR::ABL1-like ALL using RT-PCR, a targeted RNA panel, and RNA sequencing.

B-lymphoblastic leukemia with BCR::ABL1-like features (BCR::ABL1-like ALL) is a high-risk subtype of B-ALL driven by diverse kinase-activating genetic alterations. Accurate detection of these lesions is essential for molecular classification and guiding targeted therapies. We evaluated multiple RNA-based platforms to establish an optimized diagnostic strategy in a cohort enriched for suspected BCR::ABL1-like ALL. Among 340 newly diagnosed B-ALL patients, 33 harbored fluorescence in situ hybridization (FISH)-detected kinase gene abnormalities. Eighteen patients with sufficient RNA were included for multiplex RT-PCR, a targeted RNA panel, and whole-transcriptome RNA sequencing (WTS). WTS data were analyzed using two fusion workflows-DRAGEN and Arriba. Transcriptome profiles were classified using ALLCatchR based on TPM expression matrices. Across all platforms, 20 distinct genetic lesions were identified. Multiplex RT-PCR detected 4 events (20%), reflecting its restriction to primer-covered targets. The targeted RNA panel identified 9 lesions (45%); however, it showed panel-dependent limitations, including misclassification in 5 cases (28%) when partner genes were not included in the assay design. WTS captured 17 genetic lesions (85%) when DRAGEN and Arriba results were combined enabled recognition of both known and novel rearrangements. ALLCatchR assigned 14 of the 18 analyzable cases (78%) to the BCR::ABL1-like subtype, providing subtype resolution beyond structural variant identification. WTS, combined with robust fusion pipelines and transcriptomic classification, delivers the most comprehensive assessment of kinase-activating lesions in BCR::ABL1-like ALL. These results support optimized strategy in which WTS is prioritized for cases not fully resolved by FISH or targeted RNA panels, enabling more accurate molecular classification and guiding targeted therapy.