Project Summary/Abstract The recent discovery of the Ph-like subtype of childhood acute leukemia (ALL) represents a major breakthrough in the understanding and management of this disease. This subgroup accounts for approximately one quarter of all childhood high-risk ALL patients and is responsible for more than half of their deaths. The characteristic gene expression signature of the Ph-like patients allows them to be identified at diagnosis and facilitates an in depth characterization of their acquired underlying molecular abnormalities, including: gene fusions involving tyrosine kinase genes and their pathways, translocations and deletions of CRLF2, mutations of JAK kinases and insertions/deletions of IL7R. While the discovery of these fusions is still ongoing, many of these kinase events have been shown to be responsive to tyrosine kinase inhibitors (TKIs) and other targeted therapies, making their identification clinically crucial. The proposed work is both a continuation of the characterization of Ph-like cases and also the application of the Ph-like gene expression assay in clinical trials to permit the evaluation and development of targeted drug therapies for these patients. This involves the initial testing of a very large prospective cohort of diagnostic ALL samples to determine if the Ph-like signature is present. Simultaneously with the identification of the Ph-like signature, testing for the most common CRLF2 fusion is also performed as well as a determination of the likelihood of other CRLF2 rearrangements. The remaining Ph-like cases (comprising only about 10-15% of the initial cohort) will then be subjected to transcriptomic sequencing (RNA-Seq) for the rapid identification of expressed fusions, mutations and related insertions/deletions. This phase of the work will expand the discovery of new fusions and also permit the assignment of known fusions to targeted therapies. The combination of the discovery and characterization of these acquired events with full gene expression information will also allow new predictive models and assays to be developed. Within the next several years the culmination of this work is to transform the Ph-like ALL patients from the worst outcome group to the best. The precedent for this has been the advent of TKI therapy for BCR-ABL1 patients. Over the past ten years these patients have evolved from the poorest prognosis group to among the best outcomes, principally due to the administration of appropriate targeted drugs. The challenge in doing this with Ph-like patients has been the enormous diversity of gene fusions (currently more than 100) and the difficulty in diagnosing and characterizing them. The application of the gene expression assay and sequencing strategy described in this proposal address both of those issues and also allow for the targetable fusions to immediately become part of clinical trials.