Uncovering genomic complexity of PAX5 internal tandem duplication using long-read and short-read sequencing

PAX5 internal tandem duplications (PAX5-ITD) are associated with poor outcomes in childhood B-ALL and its precise genomic architecture remains unclear. In this study, we report the analysis of 26 PAX5-ITD cases using long-read and short-read sequencing. We observed an inverse correlation between the size of the duplicated region (median 23.8 kb) and the number of copies gained (median 6.0). Consistent copy number gain between DNA and RNA indicates high splicing fidelity of ITD allele. All investigated PAX5-ITDs with long-read data are in-frame. Analysis of secondary mutations within PAX5-ITD region establishes a multi-step process for PAX5-ITD formation. Concordant copy number and structural events between diagnosis and relapse samples of the same patients suggest that PAX5-ITD is an ancestral event that remains stable during clonal evolution upon therapy. Collectively, our findings highlight the genomic complexity and diverse regulatory features of PAX5-ITD formation and provide a foundation for functional investigation and precision targeting.