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INTRODUCTION: Targeted drug therapy is the Holy Grail of oncology. Through the understanding of key molecular pathways involved in tumor growth, specific inhibitors can be designed to inactivate these pathways, thereby maximizing therapeutic response. One of the most compelling examples of successful targeted therapy for a metastatic solid tumor is imatinib (Gleevec), which inhibits the KIT tyrosine kinase in gastrointestinal stromal tumors (GIST). Dramatic clinical responses are seen routinely after initiation of Gleevec therapy, but most patients eventually relapse as the tumors develop resistance to the drug. Additional specific inhibitors of the KIT signaling pathway are currently undergoing clinical trials. The aim of this study was to develop a robust, cytology-based assay to measure tumor susceptibility to target-specific small molecule inhibitors. MATERIALS AND METHODS: We created an immortal GIST cell line (GIST882), which was treated, in vitro, with the Gleevec inhibitor of KIT, and the RAD001 inhibitor of the KIT downstream signaling protein, mTOR. (The signaling pathway proteins activated by the GIST KIT mutations are sequentially: KITΟΎ ΟΎAKTΟΎ ΟΎmTORΟΎ ΟΎS6.) Treatment response was assessed in cytologic preparations (ThinPrep and cytospin) by immunocytochemical staining with antibodies to KIT, phospho-KIT, phospho-AKT, and phospho-S6. Optimization was performed to max-imize staining in the absence of inhibitor, and minimize staining in the presence of inhibitor. RESULTS: GIST882 cells demonstrated strong, robust phospho-S6 expression in the absence of inhibitor treatment. This expression was completely inhibited by treatment with upstream signaling pathway inhibitors (Gleevec and RAD001.) Other phospho-specific antibodies (phospho-AKT and Phospho-KIT) had weaker baseline reactivity in the absence of inhibitor. The accuracy of the immunocytochemical results on the cytologic preparations was validated by immunoblotting studies. CONCLUSION: Cytologic phospho-S6 staining can be used to gauge GIST response to KIT or mTOR inhibitors. This approach provides a straightforward strategy to measure biochemical responsiveness (or lack thereof) in patients receiving targeted chemotherapy and will be a useful tool for evaluating the efficacy of targeted therapies. Our study demonstrates the feasibility of cytologic methods to monitor labile biochemical responses in tumor cells during drug therapy. Such approaches will be enhanced by the development of additional activation state-specific antibodies, particularly those optimized for use in cytologic preparations.
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