Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Fusion genes are hallmarks of ALL, and they are used as biomarkers for risk stratification as well as targets for precision medicine. Hence, clinical diagnostics pursues broad and comprehensive strategies for accurate discovery of fusion genes. Currently, the gold standard methodologies for fusion gene detection are fluorescence in situ hybridization and polymerase chain reaction; these, however, lack sensitivity for the identification of new fusion genes and breakpoints. In this study, we implemented a simple operating procedure (OP) for detecting fusion genes. The OP employs RNA CaptureSeq, a versatile and effortless next-generation sequencing assay, and an in-house as well as a purpose-built bioinformatics pipeline for the subsequent data analysis. The OP was evaluated on a cohort of 89 B-cell precursor ALL (BCP-ALL) pediatric samples annotated as negative for fusion genes by the standard techniques. The OP confirmed 51 samples as negative for fusion genes, and, more importantly, it identified known (KMT2A rearrangements) as well as new fusion events (JAK2 rearrangements) in the remaining 38 investigated samples, of which 16 fusion genes had prognostic significance. Herein, we describe the OP and its deployment into routine ALL diagnostics, which will allow substantial improvements in both patient risk stratification and precision medicine.

Cancer Center Humanitas Research Hospital Humanitas University Rozzano Milan Italy

Center of Biostatistics for Clinical Epidemiology Department of Health Science University of Milano Bicocca Milan Italy

Central European Institute of Technology Masaryk University Brno Czech Republic

Centro Ricerca Tettamanti Clinica Pediatrica Università degli Studi di Milano Bicocca Fondazione MBBM Ospedale S Gerardo Monza Italy

Clinica Pediatrica Università degli Studi di Milano Bicocca Fondazione MBBM Ospedale S Gerardo Monza Italy

Department of Biology University of Bari Aldo Moro Bari Italy

Department of Hematology University Hospital Schleswig Holstein Kiel Germany

Pediatric Hematology Oncology Unit Department of Pediatrics University of Milano Bicocca MBBM Foundation ASST Monza Monza Italy

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