Application of Targeted RNA‐Sequencing in High‐Risk B‐Cell Acute Lymphoblastic Leukemia (B‐ALL): Identifying Fusions, IKZF1 Deletions, and CRLF2 Expression in an Indian Cohort

Introduction B‐cell acute lymphoblastic leukemia (B‐ALL) is genetically heterogeneous. We assessed the utility of FusionPlex ALL targeted RNA sequencing panel in detecting gene fusions and other genomic lesions in B‐ALL. Methods The high‐risk B‐ALL, negative for common recurrent gene fusions (RGF), that is, BCR::ABL1 , ETV6::RUNX1 , TCF3::PBX1 and KMT2A::AFF1 , were analysed with RNA‐based targeted sequencing 81‐gene‐panel FusionPlex ALL (IDT, USA). Multiplex ligation‐dependent probe amplification (MLPA) was used for IKZF1 deletions and flow‐cytometry for CRLF2 expression and ploidy analysis. Results Out of 32 samples, 27 were high‐risk B‐ALL cases (median age 16 (1–41) years) and 5 B‐ALL controls with known fusions for validation. The fusions were detected in 6/27 (22%) RGF‐negative B‐ALL cases; 2 with EPOR::IGH and 1 each P2RY8::IGH, PAX5::ETV6, SNX2::ABL1, IKZF1::CIITA . In addition, IKZF1 and/or PAX5 gene deletions resulting in the formation of oncogenic/novel isoforms were detected in 75% (15/20) samples positive on MLPA. Flow‐cytometry CRLF2 overexpression was noted in 60% (9/15) tested samples which correlated well with targeted RNAseq CRLF2 gene expression. Conclusion The targeted sequencing approach can help in detecting known and novel fusions in B‐ALL, novel breakpoints in the known fusions, gene deletions as oncogenic/novel isoforms and CRLF2 expression.