Targeted Locus Amplification & Next Generation Sequencing for the Detection of Recurrent and Novel Gene Fusions for Improved Treatment Decisions in Pediatric Acute Lymphoblastic Leukemia
Authors: Kuiper Roland P. , van Reijmersdal Simon V. , Simonis Marieke , Yu Jiangyan , Sonneveld Edwin , Scheijen Blanca , Boer Judith M. , Boeree Aurélie , Klous Petra , Hoogerbrugge Peter M , Yilmaz Mehmet , van Leeuwen Frank N. , Den Boer Monique L. , Splinter Erik 
Published: October 2019

Abstract
Current risk assessments for treatment of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are based on several clinical and biological criteria, including genomic alterations. Genomic profiling of BCP-ALL in the last few years has substantially extended the number of risk factors that can be used for risk stratification, including a novel entity known as BCR-ABL1 -like or Ph-like ALL. This subgroup of BCR-ABL1-like cases is characterized by the high recurrence of a diverse repertoire of novel gene fusions and mutations which frequently result in enhanced tyrosine kinase and cytokine receptor signaling [Roberts et al., NEJM 2014;371:1005-15]. Leukemia's with these alterations could potentially be targeted with appropriate tyrosine kinase inhibitors. Clinical trials with newly-diagnosed patients carrying these alterations are therefore required, but the large genomic diversity within this group of patients currently provides a major bottleneck.
Here, we describe the use of Targeted Locus Amplification (TLA), combined with deep-sequencing to detect fusion genes and sequence mutations relevant for stratification of BCP-ALL. TLA involves a strategy to selectively amplify and sequence regions >100 kb around a preselected primer pair by crosslinking of physically proximal genomic sequences [de Vree et al., Nat Biotechnol. 2014;32:1019-25]. Since TLA results in the amplification of all sequences at either end of the primer pair, TLA is highly effective in picking up structural variations including novel fusion partners. Furthermore, breakpoints can be identified from the TLA sequencing data from which targets for detection of minimal residual disease can be directly designed.
A total of 31 primer sets targeting 19 recurrently affected genes were designed and multiplexed, including the 'classical' players MLL, RUNX1, TCF3, and IKZF1, the tyrosine kinase genes ABL1, ABL2, PDGFRB, CSF1R, JAK1, JAK2, JAK3, FLT3, and TYK2, and the cytokine signaling genes CRLF2, EPOR, IL7R, TSLP, SH2B3, and IL2RB. Primer sets were chosen such that the most relevant regions were sufficiently covered. As a pilot, viable cells from 46 selected BCP-ALL samples were analysed, including 26 cases with a BCR-ABL1 -like expression profile [Den Boer et al., Lancet Oncol. 2009;10:125-34], of which 6 had a known kinase fusion. 7 Gb of aligned sequence data was obtained for each patient sample. All 21 rearrangements known to be present in these samples were detected by TLA, including rearrangements in ETV6-RUNX1 (n=5), MLL (n=4), TCF3-PBX1 (n=3), CRLF2 (n=4), EBF1-PDGFRB (n=2), BCR-ABL1 (n=1), RCSD1-ABL2 (n=1), and SSBP2 -CSF1R (n=1). For 10 of the fusions sequencing depth was sufficient to extract breakpoint-spanning sequences directly. For two cases with known JAK2 fusions with an unknown partner, the fusion gene was identified (TERF2 and BCR), as was the case for an unknown ABL1 fusion (FOXP1). New fusions were identified in 9 cases, including previously described IGH@-EPOR and TCF3-ZNF384 fusions, and novel kinase activating fusions of MAP3K19-TSLP and HDAC9-FLT3. In addition we identified deletion breakpoint fusions in IKZF1, and sequence mutations in JAK1, JAK3,and IL7R. In total, we detected gene fusions or sequence mutations affecting tyrosine kinase or cytokine receptor signaling in 16 of the 26 cases with a BCR-ABL1 -like expression profile. We conclude that TLA is an effective method for the reliable detection of sequence mutations and structural variations that are relevant for disease prognosis and/or could be targeted by approved kinase inhibiton.

Disclosures
Simonis: Cergentis BV: Employment. Klous:Cergentis BV: Employment. Yilmaz:Cergentis BV: Employment. Splinter:Cergentis BV: Employment.