Calhoun, Benjamin C.

Abstract Background: Tumor infiltrating lymphocytes (TILs) play an important, but incompletely understood role in chemotherapy response and prognosis. In breast cancer, there appear to be distinct immune responses by subtype, but most studies have used limited numbers of protein markers or bulk sequencing of RNA to characterize immune response, in which spatial organization cannot be assessed. Methods: To identify immune phenotypes of Basal-like vs. Luminal breast cancer we used the GeoMx® (NanoString) platform to perform digital spatial profiling (DSP) of immune-related proteins in tumor whole sections and tissue microarrays (TMA). Visualization of CD45, CD68, or pan-Cytokeratin by immunofluorescence was used to select regions of interest (ROIs) in formalin-fixed paraffin embedded tissue sections. 44 antibodies representing stromal markers and multiple immune cell types were applied to quantify the tumor microenvironment. Results: In whole tumor slides, immune hot spots (CD45+) had increased expression of many immune markers, suggesting a diverse and robust immune response. In epithelium-enriched areas, immune signals were also detectable and varied by subtype, with Regulatory T cell (Treg) markers (CD4, CD25, FOXP3) being higher in Basal-like vs. Luminal breast cancer. Extending these findings to TMAs with more patients (n=75), we confirmed subtype-specific immune profiles, including enrichment of Treg markers in Basal-likes. Conclusion: This work demonstrated that immune responses can be detected in epithelium-rich tissue, and that TMAs are a viable approach for obtaining important immunoprofiling data. In addition, we found that immune marker expression is associated with BC subtype, suggesting possible prognostic or targetable differences. Citation Format: Andrea Walens, Linnea T. Olsson, Xiaohua Gao, Alina M. Hamilton, Erin L. Kirk, Stephanie M. Cohen, Bentley R. Midkiff, Yongjuan Xia, Mark E. Sherman, Nana Nikolaishvili-Feinberg, Katherine A. Hoadley, Melissa A. Troester, Benjamin C. Calhoun. Protein-based immune profiles of basal-like vs luminal breast cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-090.

Abstract Background Multigene assays for breast cancer are becoming more commonly used for research, diagnostic, and treatment-selection purposes. These assays are commonly based on a single tumor sample, so this study assesses whether clinically-relevant multigene scores are subject to misclassification due to technical variation or intratumoral heterogeneity. Methods Using FFPE blocks, tumors from 37 patients were sampled at different pathologist-selected spatial locations, targeting regions with distinct histological appearance. A second pathologist assessed mitotic activity and presence of immune infiltration for each specimen. Gene expression was quantified using the PAM50 assay. Samples were classified with respect to intrinsic subtype (Luminal A, Luminal B, HER2-enriched, or Basal-like) and risk of recurrence with proliferation score (ROR-P, high vs. med/low). Euclidean distances between samples were calculated across the 50 genes, and the distribution of Euclidean distances were compared for samples that were concordant vs. discordant for each multigene classifier for PAM50 or ROR-P. Results Of the 37 tumors, 11 had samples with discordant PAM50 subtype and 7 had samples with discordant ROR-P group (75% and 83% agreement, respectively). The Euclidean distance between paired samples with discordant PAM50 intrinsic subtype or ROR-P score was significantly greater than that among concordant tumors. Samples discordant for ROR-P had more heterogeneity in histological characteristics, while discordance of PAM50 subtype call was generally not predicted by heterogeneity of these histological characteristics. Conclusion The concordance of PAM50 subtype calls and ROR-P, despite oversampling of heterogenous-appearing tumor regions, demonstrates reproducibility of the PAM50 classifier for breast cancer tumor intrinsic subtyping. However, assay sampling strategies for histologically heterogeneous tumors merit further consideration. Citation Format: Amber N. Hurson, Alina Hamilton, Linnea T. Olsson, Erin L. Kirk, Benjamin C. Calhoun, Joseph Geradts, Mark E. Sherman, Melissa A. Troester. Intratumoral heterogeneity of prognostic multigene signatures for breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-068.