eP053: Detection of rare and novel fusions in pediatric B-Lymphoblastic Leukemia (B-ALL) by capture-based transcriptome sequencing (RNA-Seq)


      Numerous newly recognized clinically significant genetic markers in B-lymphoblastic leukemia (B-ALL) that are being incorporated into the next edition of the World Health Organization (WHO) classification cannot be detected by routine karyotype and fluorescence in situ hybridization (FISH) analyses. For example, EPOR and CRLF2 rearrangements associated with the BCR-ABL1 (Philadelphia)-like subtype of B-ALL and abnormalities which define DUX4-upregulated subtype of the disease, remain undetected by standard cytogenetic evaluation. At Children’s Hospital of Los Angeles (CHLA) we incorporate Chromosomal Microarray (CMA) and a custom next-generation sequencing (NGS) panel (OncoKids) into routine clinical assessment for hematologic malignancies. However, even after this comprehensive evaluation, the primary genetic driver remains unknown in up to 15% of our B-ALL cases. We hypothesized that capture-based transcriptome sequencing (RNA-Seq) and, in selected cases, confirmatory Optical Genome Mapping (OGM), could be used to identify clinically significant oncogenic fusions missed by our routine multi-modal B-ALL testing.


      This study of archival clinical material included 53 pediatric B-ALL cases in which the genetic driver remained unknown after clinical karyotyping, FISH, CMA, and OncoKids analyses. RNA-Seq was conducted by enrichment with the Twist Comprehensive Exome capture probes set, followed by sequencing on the NextSeq or HiSeq instrument (Illumina, Inc., San Diego, CA). Data analysis for detection of abnormal gene fusions utilized a NGS analysis pipeline that incorporated Fusion Catcher, Dragen, STARfusion, and Arriba. Gene expression measurements were obtained using featureCounts. Normalized expression levels for the DUX4 gene were evaluated by comparison with B-ALL samples that were known to contain or lack oncogenic DUX4 rearrangements. Selected novel fusions detected by RNA-Seq were confirmed by OGM, a novel platform for high-resolution, genome-wide detection of copy number abnormalities and balanced chromosomal rearrangements, performed at the Bionano Genomics Service Laboratory (Bionano Genomics, San Diego, CA).


      RNA-Seq identified rare, previously described B-ALL fusions in 8/53 cases, and IGH rearrangements in 5/53 cases. In addition, RNA-Seq revealed novel fusions in 6/53 cases. Overall, confirmed or putative oncogenic drivers were identified in 19/53 (36%) B-ALLs. Recently described genetic subtypes identified in our cohort included MEF2D fusions with different partner genes (BCL9, FOXJ2, and SS18) in 4 cases, and EP300-ZNF384, TCF3-PBX1, SPTBN1-PDGFRB, and ACIN1-NUTM1 fusions in one case each. The detected IGH rearrangements were IGH-CEBPA (1 case), IGH-IL3 (1 case), and IGH-DUX4 (3 cases). The IGH-DUX4 rearrangements were confirmed by DUX4 overexpression. Finally, the novel candidate fusions in 6 cases included two JAK2 fusions with previously unreported partner genes (RBM26 and STRBP), both of which were confirmed by OGM, and PAX5-HIPK3, ETV6-MFN2, ZMYND8-PDGFRB and BACH2-ATRX fusions for which confirmation is in progress.


      In this retrospective study, RNA-Seq detected clinically significant gene fusions or IGH rearrangements in 13/53 (24.5%) of the cases which had remained negative after comprehensive clinical testing. These detected fusions define specific genetic subtypes of B-ALL and represent important prognostic markers. We also demonstrate the clinical utility of OGM, a novel technology, in confirming RNA-Seq findings. Furthermore, the assays allowed the discovery of 6 novel candidate fusions, including JAK2 fusions, which are associated with the BCR-ABL1 (Philadelphia)-like subtype of B-ALL and may predict response to treatment with JAK2 inhibitors. Our results support the diagnostic and research utility of RNA-Seq in pediatric B-ALL. Considering the heterogeneity of oncogenic fusions and rapid discovery of novel fusion partners, we propose that non-targeted transcriptome sequencing may be superior to targeted, fixed-content fusion panels in clinical B-ALL testing.