Translating pharmacodynamic biomarkers from bench to bedside: analytical validation and fit-for-purpose studies to qualify multiplex immunofluorescent assays for use on clinical core biopsy specimens.

Multiplex pharmacodynamic (PD) assays have the potential to increase sensitivity of biomarker-based reporting for new targeted agents, as well as revealing significantly more information about target and pathway activation than single-biomarker PD assays. Stringent methodology is required to ensure reliable and reproducible results. Common to all PD assays is the importance of reagent validation, assay and instrument calibration, and the determination of suitable response calibrators; however, multiplex assays, particularly those performed on paraffin specimens from tissue blocks, bring format-specific challenges adding a layer of complexity to assay development. We discuss existing multiplex approaches and the development of a multiplex immunofluorescence assay measuring DNA damage and DNA repair enzymes in response to anti-cancer therapeutics and describe how our novel method addresses known issues.