A phenotypic assay to monitor therapeutic efficacy of signal transduction inhibitors

B69

Introduction: Cancer therapy has witnessed a paradigm shift with the introduction of the first gene-specific, targeted drugs. Major successes have been observed with signal transduction inhibitors (STI) such as Gleevec targeting the oncogenic activities of tyrosine kinases. However, it has also become clear that targeted therapy will have its limitations in the natural variability of the target gene itself. One of the consequences is that such variations can lead to different treatment response among patients. This necessitates sensitive diagnostic tools to identify the appearance of resistant variants and to predict therapy response to STI.Methods: The tyrosine kinase domain from the target gene isolated from clinical specimens is either sequenced or cloned into an expression system. Phenotypic susceptibility of the given tyrosine kinase to available STI was assessed in a cell-based reporter assay.Results: One drawback of available genotypic approaches is their requirement for prior knowledge of potential effects of mutations in the gene product on drug responsiveness. For overcoming these limitations we developed a sensitive phenotyping system. We recently validated one such phenotypic platform for inhibitors of Bcr/Abl. Genetic engineering of known therapy-resistant mutants confirmed in vitro differences in drug susceptibility. A main asset of our novel phenotyping system is its high sensitivity for therapy-relevant sub-populations typical for early phases of treatment failure: We measured resistance even if resistant variants only represented less than 1% of the total target cell population. For further validation, clinical samples from a phase II trial evaluating Dasatinib in CML were analyzed using both genotyping and phenotyping methods. In several instances resistant variants were only detected with the phenotypic assay. Yet subsequent sequencing of the Bcr/abl gene product in cells surviving drug pressure during in vitro exposure revealed the presence of relevant minority mutants in the authentic samples. At the same time, this assay identified and assigned new uncharacterized mutations in other samples. Reverse genetics confirmed that the latter variants conferred Gleevec resistance.Conclusions: Our pilot data highlight the power of the phenotyping system for functionally assessing susceptibility to STI of Bcr/Abl genes isolated from specimens. Moreover, it justifies extending this phenotypic platform to other targets such as EGFR, c-Kit, Flt3, which are currently in development. Furthermore, such test will be of utility for optimizing clinical drug development and for improving patient management as patients' susceptibility at therapy onset and/or during treatment can be readily monitored.