Comprehensive genetic characterization of adult BCR::ABL1-negative B-ALL by Optical Genome Mapping and Targeted NGS

Adult B-cell precursor acute lymphoblastic leukemia (B-ALL) is genetically heterogeneous, comprising numerous subtypes. Standard methods leave many patients unclassified, and routine implementation of whole-transcriptome sequencing (WTS) or whole-genome sequencing (WGS) is limited. We evaluated a combined workflow of Optical Genome Mapping (OGM) and targeted next-generation sequencing (t-NGS) for genetic characterization of 100 adult BCR::ABL1-negative B-ALL patients enrolled in PETHEMA-LAL-19. This approach was compared with protocol genetic testing. The integration of OGM and t-NGS enabled the classification of 84% of BCR::ABL1-negative patients into 15 distinct subtypes, reducing the proportion of B-other/unclassified case from 50% to 16%. OGM was useful in identifying BCR::ABL1-like (26% of cases), associated with post-induction minimal residual disease (MRD) positivity (72% vs. 34%, p=0.0011), while t-NGS contributed to the detection of mutation-defined subtypes (PAX5 P80R and PAX5alt), correlated with MRD negativity (91% vs. 51%, p=0.012). OGM and t-NGS identified genetic patterns of certain subtypes, including a higher prevalence of IKZF1plus in BCR::ABL1-like (62% vs. 12%, p<0.001), JAK-STAT mutations only in patients with CRLF2-rearrangements and a high prevalence of PAX5-biallelic events (92% vs. 2%, p<0.001) in PAX5-subtypes. Therefore, the integration of OGM and t-NGS represents a feasible approach that enables comprehensive genetic characterization of adult BCR::ABL1-negative B-ALL, supporting its implementation in routine diagnostic workflows.