Using sample reinterrogation to guide high-parameter, spatially resolved tissue biomarker discovery.
Authors: J. Spencer Schwarz, Benton Berigan, Matthew Ingalls, Xenia Meshik, Charles Jackson, Kayla Cashion, Arne Christians, Anke Brix, Marcus Boettcher, Oliver Braubach
Journal: Journal of Clinical Oncology (2024)

e15182 Background: High-parameter, spatially resolved phenotyping can accelerate the discovery of tissue biomarkers as well as reveal emergent disease indicators like cellular neighborhood architecture. These spatial biology techniques hold high potential as diagnostic and prognostic indicators yet can be challenging to deploy due to high cost and lack of standardization. We have developed a powerful spatial biology workflow based on multiplex cyclic immunofluorescent (IF) staining using validated markers and assay kits that allows the researcher to adjust the markers included in the assay with feedback from marker expression in previous rounds. We term this sample re-interrogation. Here we present an illustration of this new workflow. Methods: Three FFPE lung biopsy specimens were repeatedly assayed for biomarker expression using CellScape™ Precise Spatial Multiplexing paired with VistaPlex™ Assay Kits. In this workflow, tissues are mounted in enclosed fluidic chambers. IF Staining and imaging were conducted on the CellScape instrument as iterative rounds. Between each imaging round, we conducted analyses on data from previously collected rounds. Conclusions drawn between staining rounds then guided decisions of whether and how to continue the experiment. Results: Each sample provided evidence for the utility of staged rounds of tissue staining, analysis, and rational assay planning. Two FFPE lung samples from the biopsy were not considered for further iterative staining after initial interrogation for consensus markers resulted in poor expression ( e.g.: CD4, CD20, CD45, pan-cytokeratin). One of these two samples was stained with markers indicative of cell death pathways and fibrosis to provide a positive explanation for initial poor staining. A third lung tissue showed punitive indication of tertiary lymphoid structure (TLS) inclusion. Further rounds of staining were conducted to iteratively explore the nature of these punitive TLS structures. Each round of additional stains was chosen based on data derived from the previous staining series following analysis and deliberation among the biomarker team. Conclusions: The data illustrate that even among tissue sections taken from the same biopsy, a specimen’s unique biomarker expression profile may require adjustments to assay parameters. Two of the FFPE sections proved unacceptable for further investigation after only a handful of stains. This finding highlights the benefit of rational reinterrogation, as the saved resources in not staining these tissues could measure in the thousands of dollars in reagents alone. The third lung tissue specimen offers an example of a tailored spatial biology assay through iterative reinterrogation. All included biomarkers were judged best suited to elucidate the next round of staining, representing an optimized and flexible spatial biology workflow.