Implementation of Validated Pharmacodynamic Assays in Multiple Laboratories: Challenges, Successes, and Limitations

Abstract

There is a “life-cycle” of pharmacodynamic (PD) biomarker assays that guides the development and clinical implementation in our laboratories. The well-recognized elements of analytical assay validation and demonstration of fitness-for-purpose of the biomarker, specimen collection, handling and assay methods are only a part of the required activities. Assay transfer across laboratories and testing on actual human clinical specimens are vital for understanding assay performance and robustness. In our experience, this patient specimen-centered approach has required assay method modifications, some unexpected, but which were critical to successful implementation in clinical trials. Additionally, dispersing assays throughout the NCI’s clinical trials network has required the development of calibrator and control materials as well as formal training courses for smooth implementation. One measure of success of this approach has been that a number of the assays developed at Frederick National Laboratory have ultimately reached the stage of commercialization, enabling wide accessibility of the PD biomarker assays by the research community.

Novel pharmacodynamic biomarkers for MYCN protein and PI3K/AKT/mTOR pathway signaling in children with neuroblastoma

“…In the pediatric setting where phase I studies may be conducted in 10 or more sites, standard operating procedures for sample collection and processing are vital to ensure that results from multiple sites are comparable (Kinders et al., 2014). Assays require thorough optimization and evaluation to ensure that they are fit‐for‐purpose and the inclusion of appropriate controls is paramount.…”

Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug—induced DNA double strand breaks from DNA double strand breaks associated with apoptosis

“…Images were extracted with a fixed size (1,000 × 1,000 pixel) from full tissue scans using Aperio ImageScope software. Images selected for extraction were derived from tumor tissue determined to be of sufficient quality as assessed via evaluation of an adjacent H & E–stained section of each specimen by a board-certified pathologist [ 29 ]. Definiens Tissue Studio software (Definiens AG, Munich, Germany) was used for quantitative analysis of all xenograft and clinical biopsy material.…”

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

“…29,30, 31 Figure 5 shows a block diagram of our workflow for our DNA damage repair image analysis project.…”

“…If the baseline variability for the biomarker is high, the assay dynamic range must be correspondingly higher. Our starting assumption, based on detailed analysis of background signal variability within and across xenografts, 30 is that greater than a 50% change in biomarker signal is required to overcome background biomarker variability. In the case of PAR, for example, high baseline biomarker variability in the xenograft models was later confirmed in a clinical trial.…”