Abstract Introduction: While cure rates exceed 80%, many children with B-ALL still relapse. Many of these patients (pts) display a Ph-like gene expression profile (GEP), but lack canonical BCR-ABL1 fusion. We have identified alternate kinase fusions in Ph-like ALL that induce cell proliferation sensitive to tyrosine kinase inhibitors (TKI) (Roberts, NEJM 2014). We report retrospective analyses of 1390 B-ALL pts, 885 NCI high risk and 505 standard risk B-ALL pts with elevated minimal residual disease. Methods: Cases were screened using an 8-gene Taqman low-density array (LDA) PCR assay to identify the Ph-like GEP (Harvey, ASH 2013). Ph-like cases with elevated CRLF2 expression were tested for CRLF2 rearrangement (CRLF2-R; P2RY8-CRLF2 by Taqman PCR on the LDA card and IGH-CRLF2 by FISH). JAK mutations in CRLF2-R cases were tested by Sanger sequencing. Ph-like cases without CRLF2-R were tested for previously identified kinase fusions involving ABL1, ABL2, CSF1R, JAK2, NTRK3, and PDGFRB by RT-PCR. Ph-like cases without detected fusions underwent RNA-sequencing, either using standard Illumina library preparation or a customized kinome capture kit (Agilent). Results: 339 (24%) pts were Ph-like. BCR-ABL1 (N = 45) and ETV6-RUNX1 (N = 11) were excluded from further analyses, as the former already receives TKI therapy and ETV6-RUNX1 ALL lacks targetable kinase fusions (unpublished). Of the remaining 283 Ph-like cases, 153 were CRLF2high (defined by CRLF2 expression levels on the LDA card). 61 (40%) had P2RY8-CRLF2 fusion, and of the remaining 91 CRLF2high cases, 56 of 69 tested had CRLF2-R (55 to IGH, 1 to an unknown partner). Thus, 117/130 (90%) CRLF2high Ph-like cases had a documented CRLF2 genomic lesion and 52 (44%) of these had a JAK mutation. Of the 130 Ph-like CRLF2low cases, 61 (47%) had a previously reported targetable TK fusion identified by RT-PCR, kinome capture or RNA sequencing. These included: 38 ABL class fusions (17 ABL1, 5 ABL2, 3 CSF1R, 13 PDGFRB) sensitive to imatinib/dasatinib; 14 JAK2 and 8 EPOR fusions sensitive to ruxolitinib; and 1 NTRK3 fusion sensitive to crizotinib. Nine cases had known fusions with new alternate breakpoints, and an additional 9 cases had fusions of novel N-terminal partners with known actionable C-terminal kinase genes. RNA sequencing identified 8 cases with IGH-EPOR fusions not previously captured by the kinome assay, indicating the cryptic and complex nature of this rearrangement. Conclusion: Almost half of Ph-like pediatric B-ALL pts lacking CRLF2-R harbor altered TKs with compelling pre-clinical data that they are likely amenable to targeted therapy using FDA-approved TKIs. The COG will start real-time screening with this algorithm in 2015 and allocate pts with ABL class fusions to treatment with chemotherapy plus dasatinib. Citation Format: Shalini C. Reshmi, Richard C. Harvey, Amy Smith, I-Ming Chen, Marc Valentine, Yu Liu, Yongjin Li, Jinghui Zhang, Kathryn G. Roberts, Ying Shao, John Easton, Debbie Payne-Turner, Meenakshi Devidas, Nyla Heerema, Andrew J. Carroll, Elizabeth A. Raetz, Michael J. Borowitz, Brent L. Wood, Anne L. Angiolillo, Michael M. Burke, Wanda L. Salzer, Patrick A. Zweidler-McKay, Karen R. Rabin, William L. Carroll, Mignon L. Loh, Stephen P. Hunger, Charles G. Mullighan, Cheryl L. Willman, Julie M. Gastier-Foster. Frequency of actionable gene fusions in patients with Philadelphia chromosome-like (Ph-like) B-acute lymphoblastic leukemia (ALL): A retrospective study from the Children's Oncology Group (COG). abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2015-4729