Abstract P4-09-10: Spatial protein and RNA expression in tumor, immune and stromal cells in BRCA1/2 mutated metastatic breast cancer
Authors: Collier Katharine A. , Miller Katherine , Li Zaibo , Westfall Jesse , Tallman David , Vater Mark , Tinoco Gabriel , Mardis Elaine , Stover Daniel 
Published: March 2023

Background: Spatial transcriptomics and proteomics are cutting-edge techniques that allow for quantification of RNA and protein expression within separate cell populations or regions of interest in a tissue biopsy specimen. We aimed to independently characterize the tumor cells and immune infiltrate in metastatic site biopsy specimens from patients with somatic or germline BRCA1/2 mutations. Methods: We performed spatial transcriptomics and proteomics using the Nanostring GeoMx Digital Spatial Profiler (DSP) on 9 formalin-fixed paraffin-embedded (FFPE) tissue biopsy specimens from 5 patients with metastatic breast cancer and a known somatic or germline BRCA1/2 mutation. Each patient had 1-3 separate metastatic site tissue biopsy specimens. Cell populations of interest were delineated with fluorescently labeled antibodies to pan-cytokeratin on tumor cells, CD45 of immune cells, and a nuclear stain. On each sample, 4-5 regions of interest (ROIs) were selected near the interface of the metastatic tumor deposit and surrounding normal tissue. Each ROI was segmented into 3 areas of interest (AOIs): pan-cytokeratin-positive tumor cells, CD45-positive immune cells, and the remaining stromal cells negative for both pan-cytokeratin and CD45. Data was background corrected and normalized with internal negative and positive internal controls. Relative abundance of immune cell subtypes was derived from CD45+ cell gene expression data using the TIMER algorithm. Results: The expression of 57 proteins and 84 RNAs was quantified in each of the 396 AOIs. Separate analysis of CD45+ cells, pan-cytokeratin cells, and stromal cell populations confirmed expected differential expression of immune, tumor and stromal markers, respectively. Protein expression of the estrogen receptor (ER) and progesterone receptor (PR) on pan-cytokeratin-positive tumor cells using the DSP correlated with pathologist reviewed immunohistochemistry (IHC) for ER and PR. Significant differential expression was found within each cell population between hormone receptor (HR) positive and negative cohorts, including enrichment for S100B in HR negative samples. Comparison will be shown for expression of 14 overlapping genes/proteins. Immune cell compartment gene expression deconvolution showed heterogeneity in the relative proportion of infiltrating immune cell subtypes between samples and also between ROIs within a sample. Changes in gene and protein expression from a patient with 3 serial biopsy specimens can be tracked with substantial heterogeneity across distinct AOIs within individual specimens and across metastatic sites.. Conclusions: Spatial transcriptomics and proteomics is feasible for concurrent, relative quantification of protein and gene expression in tumor, immune, and stromal cells in FFPE tissue biopsies from patients with metastatic breast cancer harboring germline or somatic BRCA1 or BRCA2 mutations. Heterogeneity within individual samples and across serial time points offers opportunity for methods development in the analyses and interpretation of spatial transcriptomic/proteomic data.

Citation Format: Katharine A. Collier, Katherine Miller, Zaibo Li, Jesse Westfall, David Tallman, Mark Vater, Gabriel Tinoco, Elaine Mardis, Daniel Stover. Spatial protein and RNA expression in tumor, immune and stromal cells in BRCA1/2 mutated metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-10.