De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells.

BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

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

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.

Adult systemic anaplastic large-cell lymphoma: recommendations for diagnosis and management.

Systemic anaplastic large-cell lymphomas (sALCLs) comprise a heterogeneous group of relatively rare T-cell non-Hodgkin lymphomas (NHLs) characterized by CD30 expression and other unifying pathologic features. Anaplastic lymphoma kinase (ALK) fusions are present in about 50% of cases. Pathological diagnosis can be challenging, particularly in ALK-negative cases. Though ALK-positive and ALK-negative sALCLs are similar morphologically and immunophenotypically, they are separate entities with different genetics, clinical behavior, and outcomes. Evidence-based data evaluating treatment regimens are limited as randomized controlled trials are lacking and most prospective studies are too small to draw definitive conclusions. However, recent advances in molecular biology are bringing forth much-needed knowledge in this field, and are likely to guide further targeted therapeutic development.