The application of RNA sequencing for the diagnosis and genomic classification of pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy, and
implementation of risk-adapted therapy has been instrumental in the dramatic improvements in
clinical outcomes. A key to risk-adapted therapies includes the identification of genomic features of
individual tumors, including chromosome number (for hyper- and hypodiploidy) and gene fusions,
notably ETV6-RUNX1, TCF3-PBX1 and BCR-ABL1 in B-cell ALL (B-ALL). RNA-sequencing
(RNA-seq) of large ALL cohorts has expanded the number of recurrent gene fusions recognized as
drivers in ALL, and identification of these new entities will contribute to refining ALL risk
stratification. We used RNA-seq on 126 ALL patients from our clinical service to test the utility of
including RNA-seq in standard of care diagnostic pipelines to detect gene rearrangements and
IKZF1 deletions. RNA-seq identified 86% of rearrangements detected by standard of care
diagnostics. KMT2A-rearrangements (MLL), whilst usually identified, were the most commonly
missed by RNA-seq as a result of low expression. RNA-seq identified rearrangements that were not
detected by standard of care testing in nine patients. These were found in patients who were not
classifiable using standard molecular assessment. We developed an approach to detect the
commonest IKZF1 deletion from RNA-seq data and validated this using a RQ-PCR assay. We
applied an expression classifier to identify Ph-like B-ALL patients. T-ALL proved a rich source of
novel gene fusions which have clinical implications or provide insights into disease biology. Our
experience shows that RNA-seq can be implemented within an individual clinical service to
enhance the current molecular diagnostic risk classification of ALL.EGA study EGAS00001004212