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Mutation-Independent Anaplastic Lymphoma Kinase Overexpression in Poor Prognosis Neuroblastoma Patients

¹ Pediatric Oncology Unit, ² Anatomic Pathology Department, ³ Department of Experimental Oncology, ⁴ Cristina Gandini Medical Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy; ⁵ Italian Neuroblastoma Foundation, ⁶ Translational Pediatric Oncology, National Cancer Research Institute (IST); ⁷ Laboratory of Pathology, ⁸ Division of Medical Oncology, ⁹ Laboratory of Oncology Istituto G.Gaslini, Genoa, Italy; ¹⁰ National Nanotechnology Laboratory of CNR-INFM, University of Salento, Lecce, Italy; ¹¹ Oncology Research, Cephalon Inc., West Chester, Pennsylvania; and ¹² Department of Clinical Medicine, Università di Milano-Bicocca, Monza, Italy

Requests for reprints: Lorena Passoni, Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milano, Italy. Phone: 39-02-23903240; Fax: 39-02-23902648; E-mail: lorena.passoni{at}istitutotumori.mi.it.

Key Words: Neuroblastoma • wild-type ALK • protein level of expression • kinase activation • molecular target

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK^high 12.8%, ALK^low 73%, P = 0.0035; cell death: ALK^high 56.4%, ALK^low 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs. [Cancer Res 2009;69(18):7338–46]