Comparative Analysis of Targeted RNA-Seq and Optical Genome Mapping for Detecting Gene Rearrangements in Acute Leukemia.

Background/Objectives: Gene rearrangements involving oncogenes are major drivers in acute leukemia, influencing disease classification, prognosis, and therapeutic decision-making. Targeted RNA sequencing (RNA-Seq) panels capable of detecting intergenic and intragenic fusions across multiple genes are increasingly used in diagnostic settings. However, comparative evaluation with orthogonal technologies remains limited. Material and Methods: We compared the performance of a 108-gene anchored multiplex PCR (AMP)-based RNA-Seq panel with that of Optical Genome Mapping (OGM) in 467 acute leukemia cases. The cohort included 360 cases of acute myeloid leukemia (AML), 89 B-lymphoblastic leukemia (B-ALL), 12 T-lymphoblastic leukemia (T-ALL), and 6 cases of mixed phenotype acute leukemia (MPAL). Results: Results of both methods were concordant in 175 (74.7%) of 234 detected gene/rearrangement fusions. The concordance rate varied significantly across different leukemia types, ranging from 80.2% in B-ALL to 41.7% in T-ALL (p < 0.001) OGM uniquely detected 37 of 234 (15.8%) clinically relevant rearrangements, whereas RNA-Seq exclusively identified 22 of 234 (9.4%). Enhancer-hijacking lesions, including MECOM and BCL11B rearrangements, CDK6::MNX1, and IGH rearrangements, had a markedly lower concordance (20.6%) compared with all other aberrations (93.1%) (p < 0.001). Conversely, some gene fusions arising from intrachromosomal deletions were interpreted by OGM as simple deletions rather than rearrangements or fusions. Conclusions: Targeted RNA-Seq was effective for detecting chimeric fusion transcripts and showed slightly better performance in identifying fusions resulting from deletions. However, OGM was effective for detecting enhancer-hijacking events that do not generate fusion transcripts. Both methods are complementary for the workup of acute leukemia cases.