Mutation-Independent Activation of the Anaplastic Lymphoma Kinase in Neuroblastoma.

Activating mutations of anaplastic lymphoma kinase (ALK) have been identified as important players in neuroblastoma development. Our goal was to evaluate the significance of overall ALK activation in neuroblastoma. Expression of phosphorylated ALK, ALK, and its putative ligands, pleiotrophin and midkine, was screened in 289 neuroblastomas and 56 paired normal tissues. ALK was expressed in 99% of tumors and phosphorylated in 48% of cases. Pleiotrophin and midkine were expressed in 58% and 79% of tumors, respectively. ALK activation was significantly higher in tumors than in paired normal tissues, together with ALK and midkine expression. ALK activation was largely independent of mutations and correlated with midkine expression in tumors. ALK activation in tumors was associated with favorable features, including a younger age at diagnosis, hyperdiploidy, and detection by mass screening. Antitumor activity of the ALK inhibitor TAE684 was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts. TAE684 was cytotoxic in vitro in all cell lines, especially those harboring an ALK mutation. TAE684 efficiently inhibited ALK phosphorylation in vivo in both F1174I and R1275Q xenografts but demonstrated antitumor activity only against the R1275Q xenograft. In conclusion, ALK activation occurs frequently during neuroblastoma oncogenesis, mainly through mutation-independent mechanisms. However, ALK activation is not associated with a poor outcome and is not always a driver of cell proliferation and/or survival in neuroblastoma.

The isolated rabbit heart and Purkinje fibers as models for identifying proarrhythmic liability.

INTRODUCTION: Delayed ventricular repolarization is associated with rare, but often fatal, polymorphic tachyarrhythmias named Torsades de Pointes. ICH S7B guideline recommends an integrated approach for cardiovascular preclinical evaluation of new drug candidates, including action potential assays (as a Purkinje fiber test) but also proarrhythmia models. The aim of this preliminary study was to compare the respective value of two preclinical in vitro rabbit cardiac preparations-the Purkinje fiber and the isolated perfused heart (Langendorff method)-based on effects of dofetilide, a selective IKr inhibitor. METHODS: Transmembrane action potentials from rabbit Purkinje fibers were recorded using a conventional intracellular glass microelectrode. Electrocardiograms from rabbit isolated hearts were evaluated for QRS, QT and T wave durations (Tpeak-Tend). The pacing protocol was the same for both preparations (basal rate of 80 bpm and pacing of 40, 60 and 140 bpm). Dofetilide was tested in both systems at concentrations of 1, 3 and 10 nmol/L. RESULTS: In Purkinje fibers dofetilide induced a concentration- and reverse use-dependent increase in action potential durations measured at 50 and 90% of repolarization. At 10 nmol/L, only 3/10 fibers showed early after depolarizations. In the isolated heart model, dofetilide also induced a similar concentration- and reverse use-dependent increase in QT-interval. From 3 nmol/L, major changes in T wave morphology, R-on-T extrasystoles and TdP were observed, mainly at low rate. Prior to arrhythmias, T wave shape and duration were markedly altered suggesting an increase in the heterogeneity of cardiac ventricular repolarization. CONCLUSIONS: The effects of dofetilide were comparable in the two models for delayed repolarization but the isolated heart appears to be a better predictor for arrhythmias and a unique in vitro model to assess arrhythmogenic potential of QT prolonging compounds at least when associated with IKr/hERG inhibition.