Abstract 2617: Uncovering genomic complexity of PAX5 internal tandem duplication using short-read and long-read sequencing

Abstract PAX5 is known to be frequently mutated in childhood B-cell precursor acute lymphoblastic leukemia (B-ALL) via several molecular mechanisms, including chimeric fusion, focal deletion, and point mutations. Among these, patients with PAX5 internal tandem duplication (PAX5-ITD) are reported to have a poor outcome. Unlike other common ITDs (e.g., FLT3), PAX5-ITD often involves gain of more than one copy of involved exons, and the exact nature of these alterations remain elusive. Here we aim to investigate the following questions: 1) what are the typical copy number gains in DNA and RNA among PAX5-ITD cases? 2) are PAX5-ITD always in-frame? 3) are these aberrations a result of multi-step or single-step events? Short-read whole genome sequencing (WGS) and RNA sequencing (RNA-seq) and PacBio ISO-seq, a state-of-the-art long-read sequencing method, are used in this study. By integrative analysis of WGS and RNA-seq data from a relapse cohort (n=584) and a non-relapse cohort (n=1055), we identified PAX5 mutation rate of ∼30% in relapsed patients versus that of ∼20% in non-relapsed patients. Among the various PAX5 mutation types, the PAX5-ITD mutation shows the greatest degree of enrichment (P=3e-6) in relapsed patients (n=21), compared with those who remain in remission (n=5). Copy number gains in PAX5-ITD cases ranged from 3 to 25 and involved four exon combinations within the gene: E5, E3-E5, E2-E5 and E1-E5 and concordant copy number gains were detected between diagnosis tumor and relapse tumor. We observed a negative correlation between the number of copy gains and the length of duplicated genomic regions. Furthermore, 11 out of the 26 PAX5-ITD cases exhibited multiple structural variants (SVs) and 1 case harbored 2 somatic SNV in ITD region. To investigate the transcriptional consequences of these SV events, we performed ISO-seq on 7 PAX5-ITD cases with available specimen. By mapping SVs and SNVs to full-length RNA transcripts, we observed complex sequences of events driving the formation of ultimate PAX5-ITD in 3 cases. Besides, copy number analysis on full-length RNA transcripts revealed variable copy number gains within tumors due to alternative splicing. Additionally, we identified neo-splice sites in 2 cases, indicating that the splicing process was influenced by the altered DNA context created by SVs. Finally, our data indicated that all PAX5-ITDs can transcribe in-frame mRNAs, suggesting activating nature of these mutations. These findings provide valuable insights into the genomic complexity of PAX5-ITD and highlight the diverse transcriptional and splicing outcomes due to these alterations. Citation Format: Yanling Liu, Bensheng Ju, Li Dong, Melanie Loyd, Yuan Feng, Heather Mulder, Pandurang Kolekar, Li Fan, Hanxia Li, Scott Foy, Geoff Neale, Patrick A. Brown, William L. Carroll, Mignon L. Loh, John Easton, Xiaotu Ma. Uncovering genomic complexity of PAX5 internal tandem duplication using short-read and long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2617.