Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma

// Manuela Krumbholz 1 , Wilhelm Woessmann 2 , Jakob Zierk 1 , David Seniuk 1 , Paolo Ceppi 3 , Martin Zimmermann 4 , Vijay Kumar Singh 2 , Markus Metzler 1, * and Christine Damm-Welk 2, * 1 University Hospital Erlangen, Department of Pediatrics, Erlangen, Germany 2 Justus-Liebig University, Department of Pediatric Hematology and Oncology, Giessen, Germany 3 Junior Research Group 1, Interdisciplinary Centre for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany 4 Hannover Medical School, Department of Pediatric Hematology and Oncology, Hannover, Germany * These authors have contributed equally to this work Correspondence to: Manuela Krumbholz, email: Manuela.Krumbholz@uk-erlangen.de Keywords: pediatric oncology; ALK-positive anaplastic large cell lymphoma; NPM-ALK fusion; genomic fusion sequences; minimal disease monitoring Received: February 01, 2018 Accepted: May 07, 2018 Published: May 29, 2018 ABSTRACT Nucleophosmin-anaplastic lymphoma kinase ( NPM-ALK) fusion genes resulting from the translocation t(2;5)(p23;q35) are present in almost 90% of childhood ALK-positive anaplastic large-cell lymphomas (ALCL). Detection and quantification of minimal disseminated disease (MDD) by measuring NPM-ALK fusion transcript levels in the blood provide independent prognostic parameters. Characterization of the genomic breakpoints provides insights into the pathogenesis of the translocation and allows for DNA-based minimal disease monitoring. We designed a nested multiplex PCR assay for identification and characterization of genomic NPM-ALK fusion sequences in 45 pediatric ALCL-patients, and used the sequences for quantitative MDD monitoring. Breakpoint analysis indicates the involvement of inaccurate non-homologous end joining repair mechanisms in the formation of NPM-ALK fusions. Parallel quantification of RNA and DNA levels in the cellular fraction of 45 blood samples from eight patients with NPM-ALK-positive ALCL correlated, as did cell-free circulating NPM-ALK DNA copies in the plasma fraction of 37 blood samples. With genomic NPM-ALK fusion sequence quantification, plasma samples of ALCL patients become an additional source for MRD-assessment. Parallel quantification of NPM-ALK transcripts and fusion genes in ALCL cell lines treated with the ALK kinase inhibitor crizotinib illustrates the potential value of supplementary DNA-based quantification in particular clinical settings.