摘要：

Anaplastic Large Cell Lymphoma (ALCL) represents a distinct subset of aggressive T-cell non-Hodgkin lymphoma (NHL) accounting for about 3% of adult NHL and 10 to 15% of childhood lymphomas. In the vast majority of the cases, ALCL is associated to chromosomal translocations, the most frequent being the t(2;5)(p23;q35), involving the Anaplastic Lymphoma Kinase (ALK) gene, which lead to aberrant NPM-ALK protein expression and kinase activity. It has been extensively demonstrated that aberrant NPM-ALK expression contributes to the pathogenesis of ALK-positive ALCL, as it causes cell transformation through activation of several biological pathways related to cell proliferation, cell-cycle control and apoptosis. Although ALK-positive ALCL have a rather benign prognosis when treated with standard chemotherapy, the failure rate at two years is almost 30% for most of these regimens. Notably, most of relapses occur within the first year from the start of therapy, and long-term survival for relapsed disease is less than 50%. Aberrant ALK activity is one of the major oncogenic events not only in ALK-positive ALCL, but also in neuroblastoma, non-small cell lung cancer (NSCLC) and inflammatory myofibroblastic tumour (IMT) bearing ALK activating mutations/rearrangements, and the inhibition of ALK kinase activity was proven to substantially reduce cancer cell proliferation and invasiveness both in vitro and in vivo. Successful clinical experience with crizotinib further support the concept of ALK-specific inhibition as a valuable treatment strategy in ALK-positive ALCL, as well as in other ALK-addicted tumours. However, similarly to other inhibitors selectively targeting oncogenic kinases, data on relapse to crizotinib due to newly acquired secondary mutations were reported. In this context, although a robust clinical response of ALCL patients to an ALK inhibitor is expected, some of those patients are also anticipated to develop resistance, making the knowledge of NPM-ALK kinase domain (KD) mutational status a valuable and mandatory information to the rational design of ALK-targeted therapies. To detect somatic tumour mutations with potential utility for predicting treatment response in ALK-positive ALCL patients, we performed ultra-deep sequencing analysis on ALK exons 22-25, corresponding to the entire KD coding region, in 37 ALCL pediatric patients. Two low frequent point mutations were identified in two distinct cases, corresponding to the R1275Q and R1231Q amino acid changes. The R1275Q mutation has been already reported as one of the most frequent activating mutations in neuroblastoma, while the R1231Q amino acid substitution represents a novel ALK point mutation, which to our knowledge has never been reported neither in ALK receptor nor in other ALK-translocated kinases. The molecular implications of R1275Q and R1231Q point substitutions on NPM-ALK function and sensitivity to ALK-specific inhibition are still under our investigation. In addition to point mutation, oncokinase alternative spliced transcripts have been previously reported in patients with Bcr-Abl positive chronic myeloid leukemia and more recently ALK receptor isoforms were described in neuroblastoma. To our knowledge, however, NPM-ALK alternative splicing events have never been described. For the first time, we identified and characterized 9 NPM-ALK INDEL mutations, resulting from KD whole exons skipping or alternative canonical splicing sites recognition. To investigate the effect of INDEL mutations on the structure and activity of NPM-ALK, we performed molecular homology modelling and in vitro functional analysis. While all these mutants were shown to be kinase dead, we demonstrated that, when coespressed with wild-type NPM-ALK, these INDELs do interact with wild-type monomers and are likely to inhibit ALK kinase activity and increase sensitivity to treatment with crizotinib. This work demonstrates that NPM-ALK KD point mutations are extremely rare in newly diagnosed ALCL patients, but positive selection of mutated cells could not be excluded in case of an ALK-t

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