Church, S.

Abstract Background: Multiple studies have confirmed the central role of preexisting immune response measured by stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). Emerging studies showed that not only the number of TILs but also the location of TILs is important. There are 3 distinct immune architectures described based on the amount and locations of TILs, namely immune enriched (IN), immune excluded (IE), and immune desert (ID). Here we evaluated outcomes and characteristics associated with each immune landscape. Methods: NanoString Digital Spatial Profiling (DSP) and CosMx, a spatial multi-omics single-cell imaging platform, were performed in 75 samples from the Mayo Clinic (MC) TNBC cohort (Leon-Ferre BCRT 2018). NanoString IO360 was performed in 114 samples from the FinXX trial ( NCT00114816) . Firstly, tumors with sTIL quantified by H&E ≤ 30% were classified as ID. The rest of the tumors with high sTIL > 30% were categorized according to the intratumoral CD8 protein expression by DSP, with IE having intratumoral CD8 in the lower median and IN having intratumoral CD8 in the upper median. Chi-square test, gene set enrichment, Cox regression, and Kaplan-Meier analysis were used. Differential expression listed as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: ID is associated with low Ki67 < 5% (23.3% vs. 9.6% ID, p 0.02) as well as apocrine (11/13, 84.6%) and metaplastic histology (10/12, 83%). In both univariate and multivariate analysis, patients with IN had significantly improved recurrence-free survival (RFS) compared to those with ID (HR 0.37, 95%CI 0.18-0.74, p 0.005). Despite having high sTILs, IE had poor outcomes similar to ID (HR 0.84, 95%CI 0.36-1.98). Strikingly, we identified that IE patients had significantly lower plasmacytoid dendritic cells (pDCs) compared to IN (mean 0 vs. 0.26/100 tumor cells, 95%CI 0.08-0.43 , p 0.01). Using Gene Set Enrichment Analysis to evaluate differential hallmarks between IN and IE, we identified IF Nɑ and IFNγ (FDR < 0.001) responses as significantly enriched in IN group, consistent wi th the function of pDCs, which are a subset of dendritic cells specialized in secreting high levels of type I interferon. To validate this finding, we further evaluated the 11 leading edge gene IFNɑ signature in the FinXX trial. A high IFNɑ signature score was associated with significantly improved outcomes in the FinXX trial (HR 0.21, 95%CI 0.09-0.51 , p < 0.001). Similar findings were observed using Kaplan-Meier analysis in the FinXX trial with significantly improved RFS (p 0.0006) and overall survival (p 0.0001) in patients with high IFNɑ signature scores. Furthermore, we evaluated the differential gene expression unique to IN tumors in the Mayo cohort. Expressions of MHC class I and class II in tumor cells, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DPA1, and HLA-E, were associated with IN and significantly improved outcomes (p < 0.05) compared to ID and IE. Conclusions: Highlighting the importance of spatial context, we identified that patients with IE tumors had poor outcomes despite having high TILs. Moreover, u sing an in-depth analysis with spatially defined context, we identified the central role of pDC and the significance of IF Nɑ in TNBC. Support: Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17) W81XWH-15-1-0292, P50CA015083, R35CA253187 Citation Format: Saranya Chumsri, Yi Liu, Yaohua Ma, Jodi Carter, Mark Gregory, Sarah Church, Jason Reeves, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith Perez, Roberto Leon-Ferre, David Hillman, Judy Boughey, James Ingle, Krishna Kalari, Fergus Couch, Matthew Goetz, Keith Knutson, E. Thompson. The spatially resolved single-cell atlas of the tumor immune architecture revealed the central role of IFN-alpha and plasmacytoid dendritic cells in triple-negative breast cancer in the Mayo Clinic cohort and FinXX trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-03.

Abstract 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>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.

Breast cancer is a heterogenous disease with variability in tumor cells and in the surrounding tumor microenvironment (TME). Understanding the molecular diversity in breast cancer is critical for improving prediction of therapeutic response and prognostication. High-plex spatial profiling of tumors enables characterization of heterogeneity in the breast TME, which can holistically illuminate the biology of tumor growth, dissemination and, ultimately, response to therapy. The GeoMx Digital Spatial Profiler (DSP) enables researchers to spatially resolve and quantify proteins and RNA transcripts from tissue sections. The platform is compatible with both formalin-fixed paraffin-embedded and frozen tissues. RNA profiling was developed at the whole transcriptome level for human and mouse samples and protein profiling of 100-plex for human samples. Tissue can be optically segmented for analysis of regions of interest or cell populations to study biology-directed tissue characterization. The GeoMx Breast Cancer Consortium (GBCC) is composed of breast cancer researchers who are developing innovative approaches for spatial profiling to accelerate biomarker discovery. Here, the GBCC presents best practices for GeoMx profiling to promote the collection of high-quality data, optimization of data analysis and integration of datasets to advance collaboration and meta-analyses. Although the capabilities of the platform are presented in the context of breast cancer research, they can be generalized to a variety of other tumor types that are characterized by high heterogeneity.