Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Fusion genes are hallmarks of ALL, and they are used as biomarkers for risk stratification as well as targets for precision medicine. Hence, clinical diagnostics pursues broad and comprehensive strategies for accurate discovery of fusion genes. Currently, the gold standard methodologies for fusion gene detection are fluorescence in situ hybridization and polymerase chain reaction; these, however, lack sensitivity for the identification of new fusion genes and breakpoints. In this study, we implemented a simple operating procedure (OP) for detecting fusion genes. The OP employs RNA CaptureSeq, a versatile and effortless next-generation sequencing assay, and an in-house as well as a purpose-built bioinformatics pipeline for the subsequent data analysis. The OP was evaluated on a cohort of 89 B-cell precursor ALL (BCP-ALL) pediatric samples annotated as negative for fusion genes by the standard techniques. The OP confirmed 51 samples as negative for fusion genes, and, more importantly, it identified known (KMT2A rearrangements) as well as new fusion events (JAK2 rearrangements) in the remaining 38 investigated samples, of which 16 fusion genes had prognostic significance. Herein, we describe the OP and its deployment into routine ALL diagnostics, which will allow substantial improvements in both patient risk stratification and precision medicine.

Cancer Center Humanitas Research Hospital Humanitas University Rozzano Milan Italy

Center of Biostatistics for Clinical Epidemiology Department of Health Science University of Milano Bicocca Milan Italy

Central European Institute of Technology Masaryk University Brno Czech Republic

Centro Ricerca Tettamanti Clinica Pediatrica Università degli Studi di Milano Bicocca Fondazione MBBM Ospedale S Gerardo Monza Italy

Clinica Pediatrica Università degli Studi di Milano Bicocca Fondazione MBBM Ospedale S Gerardo Monza Italy

Department of Biology University of Bari Aldo Moro Bari Italy

Department of Hematology University Hospital Schleswig Holstein Kiel Germany

Pediatric Hematology Oncology Unit Department of Pediatrics University of Milano Bicocca MBBM Foundation ASST Monza Monza Italy

Zobrazit více v PubMed

Inaba H, Greaves M, Mullighan CG. Acute lymphoblastic leukaemia. Lancet. 2013;381:1943–1955. doi:10.1016/S0140-6736(12)62187-4. PubMed PMC

Nguyen K, Devidas M, Cheng S-C, et al. Factors influencing survival after relapse from acute lymphoblastic leukemia: a Children's Oncology Group study. Leukemia. 2008;22:2142–2150. doi:10.1038/leu.2008.251. PubMed PMC

Hunger SP, Mullighan CG. Redefining ALL classification: toward detecting high-risk ALL and implementing precision medicine. Blood. 2015;125:3977–3987. doi:10.1182/blood -2015- 02-580043. PubMed PMC

Iacobucci I, Mullighan CG. Genetic basis of acute lymphoblastic leukemia. J Clin Oncol. 2017;35:975–983. doi:10.1200/JCO.2016.70.7836. PubMed PMC

Harrison CJ. Cytogenetics of paediatric and adolescent acute lymphoblastic leukaemia. Br J Haematol. 2009;144:147–156. doi:10.1111/j.1365-2141.2008.07417.x. PubMed

Felice MS, Gallego MS, Alonso CN, et al. Prognostic impact of t(1;19)/TCF3-PBX1 in childhood acute lymphoblastic leukemia in the context of Berlin-Frankfurt-Münster-based protocols. Leuk Lymphoma. 2011;52:1215–1221. doi:10.3109/10428194.2011.565436. PubMed

Winters AC, Bernt KM. MLL-rearranged leukemias-an update on science and clinical approaches. Front Pediatr. 2017;5:4.doi:10.3389/fped.2017.00004. PubMed PMC

Harvey RC, Mullighan CG, Chen I-M, et al. Rearrangement of CRLF2 is associated with mutation of JAK kinases, alteration of IKZF1, Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute lymphoblastic leukemia. Blood. 2010;115:5312–5321. doi:10.1182/blood -2009- 09-245944. PubMed PMC

Russell LJ, Capasso M, Vater I, et al. Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia. Blood. 2009;114:2688–2698. doi:10.1182/blood-2009-03-208397. PubMed

Pui C-H, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371:1030–1043. doi:10.1016/S0140-6736(08)60457-2. PubMed

Stam RW. MLL-AF4 driven leukemogenesis: what are we missing? Cell Res. 2012;22:948–949. doi:10.1038/cr.2012.16. PubMed PMC

Iqbal N, Iqbal N. Imatinib: a breakthrough of targeted therapy in cancer. Chemother Res Pract. 2014;2014:357027.doi:10.1155/2014/357027. PubMed PMC

Nowell PC, Hungerford DA. Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst. 1960;25:85–109. http://www.ncbi.nlm.nih.gov/pubmed/14427847. Accessed February 19, 2019. PubMed

Iijima-Yamashita Y, Matsuo H, Yamada M, et al. Multiplex fusion gene testing in pediatric acute myeloid leukemia. Pediatr Int. 2018;60:47–51. doi:10.1111/ped.13451. PubMed

Mertens F, Johansson B, Fioretos T, et al. The emerging complexity of gene fusions in cancer. Nat Rev Cancer. 2015;15:371–381. doi:10.1038/nrc3947. PubMed

Bacher U, Shumilov E, Flach J, et al. Challenges in the introduction of next-generation sequencing (NGS) for diagnostics of myeloid malignancies into clinical routine use. Blood Cancer J. 2018;8:113.doi:10.1038/s41408-018-0148-6. PubMed PMC

Kumar S, Vo AD, Qin F, et al. Comparative assessment of methods for the fusion transcripts detection from RNA-Seq data. Sci Rep. 2016;6:21597.doi:10.1038/srep21597. PubMed PMC

Mercer TR, Clark MB, Crawford J, et al. Targeted sequencing for gene discovery and quantification using RNA CaptureSeq. Nat Protoc. 2014;9:989–1009. doi:10.1038/nprot.2014.058. PubMed

Clark MB, Mercer TR, Bussotti G, et al. Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing. Nat Methods. 2015;12:339–342. doi:10.1038/nmeth.3321. PubMed

Conter V, Bartram CR, Valsecchi MG, et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. Blood. 2010;115:3206–3214. doi:10.1182/blood -2009- 10-248146. PubMed

Haas B, Dobin A, Stransky N, et al. STAR-fusion: fast and accurate fusion transcript detection from RNA-seq. bioRxiv. 2017;120295.10.1101/120295. DOI

Winters JL, Davila JI, McDonald AM, et al. Development and verification of an RNA sequencing (RNA-Seq) assay for the detection of gene fusions in tumors. J Mol Diagn. 2018;20:495–511. doi:10.1016/J.JMOLDX.2018.03.007. PubMed

GTEx Consortium TGte The genotype-tissue expression (GTEx) project. Nat Genet. 2013;45:580–585. doi:10.1038/ng.2653. PubMed PMC

van der Linden MH, Valsecchi MG, De Lorenzo P, et al. Outcome of congenital acute lymphoblastic leukemia treated on the Interfant-99 protocol. Blood. 2009;114:3764–3768. doi:10.1182/blood -2009- 02-204214. PubMed

Pieters R, Schrappe M, De Lorenzo P, et al. A treatment protocol for infants younger than 1 year with acute lymphoblastic leukaemia (Interfant-99): an observational study and a multicentre randomised trial. Lancet. 2007;370:240–250. doi:10.1016/S0140-6736(07)61126-X. PubMed

Roberts KG, Morin RD, Zhang J, et al. Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia. Cancer Cell. 2012;22:153–166. doi:10.1016/j.ccr.2012.06.005. PubMed PMC

Puig-Oliveras A, Revilla M, Castelló A, et al. Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat. Sci Rep. 2016;6:31803.doi:10.1038/srep31803. PubMed PMC

Babiceanu M, Qin F, Xie Z, et al. Recurrent chimeric fusion RNAs in non-cancer tissues and cells. Nucleic Acids Res. 2016;44:2859–2872. doi:10.1093/nar/gkw032. PubMed PMC

Nicorici D, Şatalan M, Edgren H, et al. FusionCatcher – a tool for finding somatic fusion genes in paired-end RNA-sequencing data. bioRxiv. 2014;011650.doi:10.1101/011650.

Kim RN, Kim A, Choi S-H, et al. Novel mechanism of conjoined gene formation in the human genome. Funct Integr Genomics. 2012;12:45–61. doi:10.1007/s10142-011-0260-1. PubMed

Prakash T, Sharma VK, Adati N, et al. Expression of conjoined genes: another mechanism for gene regulation in eukaryotes. PLoS One. 2010;5:e13284.doi:10.1371/journal.pone.0013284. PubMed PMC

Mullighan CG, Su X, Zhang J, et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med. 2009;360:470–480. doi:10.1056/NEJMoa0808253. PubMed PMC

Sevov M, Bunikis I, Häggqvist S, et al. Targeted RNA sequencing assay efficiently identifies cryptic KMT2A (MLL)-fusions in acute leukemia patients. Blood. 2014;124: http://www.bloodjournal.org/content/124/21/2406?sso-checked=true. Accessed March 2, 2019.

Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25:1105–1111. doi:10.1093/bioinformatics/btp120. PubMed PMC

Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi:10.1093/bioinformatics/bts635. PubMed PMC

Kim D, Salzberg SL. TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol. 2011;12:R72.doi:10.1186/gb -2011- 12-8-r72. PubMed PMC

Harrow J, Frankish A, Gonzalez JM, et al. GENCODE: The reference human genome annotation for The ENCODE Project. Genome Res. 2012;22:1760–1774. doi:10.1101/gr.135350.111. PubMed PMC

Langmead B, Trapnell C, Pop M, et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25.doi:10.1186/gb-2009-10-3-r25. PubMed PMC

Chomzynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem. 1987;162:156–159. doi:10.1006/abio.1987.9999. PubMed

Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics. 2010;26:589–595. doi:10.1093/bioinformatics/btp698. PubMed PMC

Li H, Handsaker B, Wysoker A, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi:10.1093/bioinformatics/btp352. PubMed PMC

Bankevich A, Nurk S, Antipov D, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–477. doi:10.1089/cmb.2012.0021. PubMed PMC

Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26:841–842. doi:10.1093/bioinformatics/btq033. PubMed PMC

Panigrahi P, Jere A, Anamika K. FusionHub: a unified web platform for annotation and visualization of gene fusion events in human cancer. Kumar-Sinha C, ed. PLoS One. 2018;13:e0196588.doi:10.1371/journal.pone.0196588. PubMed PMC

Potential and pitfalls of whole transcriptome-based immunogenetic marker identification in acute lymphoblastic leukemia; a EuroMRD and EuroClonality-NGS Working Group study

High EVI1 Expression due to NRIP1/EVI1 Fusion in Therapy-related Acute Myeloid Leukemia: Description of the First Pediatric Case