Scintillation proximity assays (SPAs) have become a powerful tool for high-throughput screening (HTS) because they can measure the activity and binding of very diverse classes of drug targets. By applying the basic principles of ligand-receptor binding and enzyme kinetics, it is possible to build a large variety of miniaturized, high-throughput assays and screen millions of compounds. SPAs are enabled by the diversity of radiolabeled molecules and affinity tags that are commercially available. These synthetic radiotracers allow for minimal disturbance of the natural binding interactions. This article will present a comprehensive review of the technique and provide detailed information on its applications related to HTS, highlighting the major uses and giving some suggestions for future research.

Activating mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma (NB), a cancer of the peripheral nervous system, and are thought to be the primary mechanism of oncogenic activation of this receptor in this pediatric neoplasm. To address the possibility that ALK activation may occur through genomic rearrangements as detected in other cancers, we first took advantage of high-resolution arraycomparative genomic hybridization to search for ALK rearrangements in NB samples. Using complementary experiments by capture/paired-end sequencing and FISH experiments, various types of rearrangements were fully characterized, including partial gains or amplifications, in several NB cell lines and primary tumors. In the CLB-Bar cell line, we described a genomic rearrangement associated with an amplification of the ALK locus, leading to the expression of a 170 kDa protein lacking part of the extracellular domain encoded by exons 4 to 11, named ALK D4-11 . Analysis of genomic DNA from the tumor at diagnosis and relapse revealed that the ALK gene was amplified at diagnosis but that the rearranged ALK allele was observed at the relapse stage only, suggesting that it may be implicated in tumor aggressiveness. Consistently, oncogenic and tumorigenic properties of the ALK D4-11 variant were shown after stable expression in NIH3T3 cells. Moreover, we documented an increased constitutive kinase activity of this variant, as well as an impaired maturation and retention into intracellular compartments. These results indicate that genomic rearrangements constitute an alternative mechanism to ALK point mutations resulting in receptor activation. Cancer Res; 73(1); 195-204. Ó2012 AACR.