Guidelines for processing and storing liquid biopsy samples for functional assays in research and clinical testing.

PROJECT SUMMARY The World Health Organization predicts that cancer diagnoses will increase to 22 million per year within the next two decades, with a corresponding 70% rise in cancer-related deaths. Liquid biopsy-based functional tests using blood and bone marrow will be crucial for advancing cancer precision medicine and complementing molecular genetic evaluation. Multiparameter high throughput flow and imaging platforms offer enhanced sensitivity for capture of clinically important cellular processes, and are being developed to measure therapeutic modulation of target protein signaling pathways. However, the success of these novel assays will depend on uniform standards for processing liquid biopsies across academic institutions, hospitals, and commercial entities. Currently, there is a lack of data on how pre-analytical factors, involved in the collection, and storage/retrieval of liquid specimens impact functional protein-based and live cell-dependent functional assay readouts. Therefore, pre-analytical data is critically needed to standardize workflows and support reliable clinical assay results and interpretation. Our group at the Knight Cancer Institute has collected and analyzed blood and bone marrow specimens from over 2,500 leukemia patients, resulting in the world's largest functional genomic dataset of acute myeloid leukemia (AML) patient samples. Although basic SOPs for processing and storing leukemia samples for use in functional assays have been deployed, evaluation of the impact of pre-analytical variability on assay results has not been performed. The goal of this U01 project is to characterize the pre-analytical steps involved in blood and bone marrow biopsy sample handling and storage/retrieval for functional assay reporting. The project focuses on the downstream impact of pre-analytical sample handling variability in novel, clinically relevant functional assays, namely: High Throughput-Drug Sensitivity (HT-DS), Multi-parameter High Throughput Flow (MHT-flow) and Single Cell-Multiplexed Protein Imaging (SC-MPI) assays. Our project aims to establish pre-analytical sample processing guidelines to enable maximal reproducibility for functional testing outcomes, and to evaluate the feasibility and impact of applying these guidelines in collaboration with other organizations. To that end, our proposal includes implementation and evaluation of optimized SOPs by a collaborative, interdisciplinary network that integrates clinical researchers with academic and private sector scientists. Overall, the goal is to establish pre-analytical workflow guidelines that optimize phenotypic cell identity and protein-based readouts for functional platform tests that are currently being used in drug therapeutic development and precision medicine clinical trials. The results of this work will help establish well-defined workflows critical for the successful and uniform adoption of functional platform testing across national clinical sites, as well as industry, and will broadly impact the growing number of new functional tests in development and clinical translation in areas such as immuno-oncology, early detection, and measurable residual disease.