Transcriptional and Mutational Profiling of B-Other Acute Lymphoblastic Leukemia for Improved Diagnostics

SIMPLE SUMMARY: Pediatric acute lymphoblastic leukemia is the most common malignancy in children. Based on the genetic characteristics of the tumor, patients are risk-stratified and treated with different treatment intensities. However, in a proportion of cases, known as B-other, no genetic alterations relevant for risk stratification are found with routine diagnostic procedures. In this study, we performed RNA sequencing, a comprehensive and cutting-edge method, of 185 children with B-other leukemia and analyzed gene fusions, expression profiles and mutations. Furthermore, we validated our findings using commonly used diagnostic techniques. Our results identified a subgroup of cases clustering with known leukemia subtypes, e.g., DUX4-positive, and subgroups characterized by mutations in PAX5 and IKZF1, resulting in more cases being assigned to a defined subgroup. Moreover, we identified new fusion partners of ETV6, IKZF1 and PAX5. Our data demonstrate the applicability and technical considerations for the use of RNA sequencing to personalize genetic diagnostics in pediatric leukemia. ABSTRACT: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common cancer in children, and significant progress has been made in diagnostics and the treatment of this disease based on the subtypes of BCP-ALL. However, in a large proportion of cases (B-other), recurrent BCP-ALL-associated genomic alterations remain unidentifiable by current diagnostic procedures. In this study, we performed RNA sequencing and analyzed gene fusions, expression profiles, and mutations in diagnostic samples of 185 children with BCP-ALL. Gene expression clustering showed that a subset of B-other samples partially clusters with some of the known subgroups, particularly DUX4-positive. Mutation analysis coupled with gene expression profiling revealed the presence of distinctive BCP-ALL subgroups, characterized by the presence of mutations in known ALL driver genes, e.g., PAX5 and IKZF1. Moreover, we identified novel fusion partners of lymphoid lineage transcriptional factors ETV6, IKZF1 and PAX5. In addition, we report on low blast count detection thresholds and show that the use of EDTA tubes for sample collection does not have adverse effects on sequencing and downstream analysis. Taken together, our findings demonstrate the applicability of whole-transcriptome sequencing for personalized diagnostics in pediatric ALL, including tentative classification of the B-other cases that are difficult to diagnose using conventional methods.