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Role of ALK activation in the development and maintenance of the neoplastic phenotype in neuroblastoma.

10008 Background: Activating mutations of the Anaplastic Lymphoma Kinase (ALK) receptor could be responsible for most familial neuroblastoma cases and for up to 15% of somatic cases. The objective of the present study was to further investigate the role of ALK activation in neuroblastoma.


METHODS
Tissue microarrays were constructed containing 101 primary tumors and 56 paired normal tissues. Sections were immunostained with anti-ALK or anti-P-ALK antibodies, and with antibodies directed against the ALK ligands: PTN (Pleiotrophin) or MDK (Midkine). The Wilcoxon signed rank test was applied for comparison of paired data. Associations with prognostic factors were analyzed using t-tests. Effects of the ALK inhibitor TAE684 (Novartis) on cell proliferation and signaling was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts.


RESULTS
ALK was expressed in about 100% of tumors and normal tissues, while phospho-ALK was detected in 5% of normal tissues and 50% of tumors. Sequencing of the kinase domain of ALK showed that its phosphorylation was largely independent of mutations and we found that MDK and PTN ligands were expressed in 66% and 50% of tumors, respectively. Interestingly, ALK, P-ALK, and MDK were expressed at higher levels in tumors as compared with paired normal tissues (p < 0.0001), while PTN showed an inverse tendency, being more expressed in normal tissues (p = 0.07). In tumors, P-ALK was associated with good-prognosis factors, including favorable stages (p = 0.01), absence of MYCN amplification (p = 0.05) and a younger age at diagnosis (p = 0.03). Inhibition of cell proliferation by TAE684 was detectible in all neuroblastoma cell lines, regardless of ALK status. However, TAE684 failed to demonstrate antitumor activity in advanced stage neuroblastoma xenografts expressing either a wild-type or a mutated ALK. Interestingly, ALK pathway activation (P-STAT3, P-AKT) was weak or barely detectible in these xenografts.


CONCLUSIONS
ALK activation occurs during neuroblastoma oncogenesis, along with a concomitant switch between the expressions of PTN and MDK. However, ALK may not be a relevant therapeutic target since in vivo inhibition showed no antitumor activity. [Table: see text].