Abstract 3484: Development of a custom high-plex GeoMx digital spatial profiler breast cancer protein biomarker assay
Authors: Corless Christopher , Bridgeman Amber , Lee Jinho , Geiss Gary , Warren Sarah , Church Sarah , Beechem Joseph M. , Thompson E Aubrey , Carter Jodi 
Published: June 2022

The goal of this study was to develop a high-plex assay to simultaneously quantitate 27 established and novel breast cancer (BC)-related, immune protein and phosphoprotein biomarkers using the GeoMx® Digital Spatial Profiler (DSP). The custom assay performance was compared to standard clinical BC biomarker assays (e.g.ER, PR, HER2, Ki67) across the spectrum of BC subtypes and in multiple laboratories. Commercially available antibodies to 27 BC-related protein biomarkers, including ER, PR, HER2, Ki-67, AR, immune-related (e.g. PD-L1) and several cell cycle/proliferation markers were oligonucleotide-tagged, and verified by immunohistochemistry for performance against untagged antibodies. The tagged antibodies were combined with 3 isotype controls and 2 housekeeping proteins into a custom BC high-plex assay for DSP. Confirmation of target specificity was done on a custom tissue microarray (TMA) (Run control) composed of (un)treated cancer cell lines and normal tissues. For clinical BC samples, each biopsy had 4x600 µm regions of interest segmented into pan-Cytokeratin+ tumoral epithelium and pan-Cytokeratin- adjacent stroma segments. With targeted UV light, oligonucleotides were collected from each segment sequentially and quantitated with nCounter. Raw counts were geomean normalized for analysis. Intra-site and inter-site assay reproducibility was assessed with the TMA and serial FFPE sections from BC biopsies. Quantitation of proteins showed high reproducibility within sites (e.g. Run 1 v. Run 2, p&gt;0.05 for all protein targets) and the high-plex custom assay showed good concordance between 3 participating laboratories. GeoMx DSP-derived protein data correlated well with orthogonal methodologies performed on a sample subset including immunohistochemistry, reverse phase protein array, DNA and RNA-sequencing. For example, DSP protein counts for p53 protein were higher in tumors with TP53 point mutation than those with truncating TP53 alterations. Expanded sample testing is underway with 120 well-characterized breast cancer biopsies of various histologies, immune subtypes (e.g. TIL scores) and clinical biomarker phenotypes (e.g. ER+/HER2-/Ki-67 high BC; ER+/HER2-/Ki-67 low BC; HER2+ BC; ER-/PR-/HER2- BC).Our preliminary data demonstrate that this custom high-plex BC assay can quantitate protein biomarkers across a wide dynamic range with high intra-lab and inter-lab reproducibility. The assay requirement of a single 5-µm tissue section facilitates complex biomarker profiling in biopsies with limited material. The custom assay alone or in combination with other targeted DSP protein modules can simultaneously interrogate the biomarker profile, immune-based and other drug target profile, and immune microenvironment of BC specimens, providing a novel approach for actionable tumor subtyping. For research use only. Not for use in diagnostic procedures.

Citation Format: Christopher Corless, Amber Bridgeman, Jinho Lee, Gary Geiss, Sarah Warren, Sarah Church, Joseph M. Beechem, E Aubrey Thompson, Jodi Carter. Development of a custom high-plex GeoMx digital spatial profiler breast cancer protein biomarker assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3484.