Abstract A15: Immune-related changes in breast cancer tumor evolution
Authors: Alcazar Carlos R. Gil del , Huh SungJin , Ekram Muhammad B. , Trinh Anne , Liu Lin L. , Beca Francisco , Xiaoyuan Zi , Kwak Misuk , Bergholtz Helga , Su Ying , Ding Lina , Ding Lina , Russnes Hege G. , Richardson Andrea L. , Babski Kirsten , Kim Elizabeth Min Hui , McDonnell Charles H. , Wagner Jon , Rowberry Ron , Freeman Gordon , Dillon Deborah , Sorlie Therese , Coussens Lisa M. , Garber Judy E. , Fan Rong , Bobolis Kristie , Jeong Joon , Park So Yeon , Michor Franziska , Polyak Kornelia 
Published: February 2019

Abstract
Immunotherapy is a highly promising therapeutic option in metastatic disease albeit only in a subset of patients possibly due to heterogeneity in the mechanisms by which tumors escape immune surveillance. Immune cells shape tumor evolution directly (e.g., anti-tumor immune response) and indirectly (e.g., changing the microenvironment) by selecting for cancer cells with certain properties. We hypothesized that the in situ (DCIS) to invasive ductal carcinoma (IDC) transition is a critical tumor progression step for immune escape in breast cancer that defines subsequent tumor evolution. In DCIS, cancer cells are physically separated from the stroma by the basement membrane and myoepithelial cell layer, and tumor-infiltrating leukocytes are rarely detected in direct contact with cancer cells. In contrast, in IDC, cancer cells and leukocytes are intermingled, thus, only cancer cells that can survive in this environment will play a role in disease progression. To dissect mechanisms of immune escape in breast cancer, we first analyzed the composition of leukocytes in normal breast tissues, DCIS, and IDC by polychromatic FACS. We found that DCIS and IDC contained significantly higher numbers of leukocytes, compared to normal breast, whereas in normal tissues more leukocytes were in the stromal than in the epithelial fraction. We also observed significant differences in the relative frequencies of several CD45+ cell types including increased neutrophils and decreased CD8+/CD4+ T cell ratios in tumors compared to normal stroma. Next, we analyzed the gene expression profiles of CD45+CD3+ T cells and found gene set enrichment of cytotoxic cells in DCIS including CD8+ T cells and NKT cells when compared to IDC. Conversely, we found enrichment for gene sets corresponding to regulatory T cells in IDC when compared to DCIS. Overall this suggested that DCIS had a more activated immune environment and IDC a more suppressed immune environment. We further explored this result by immunofluorescence (IF) and found fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including a set of matched DCIS and locally recurrent IDC tissues. We also found that the TCR clonotype was more diverse in DCIS than in normal breast and IDCs. Interestingly we detected a few relatively frequent clones that were shared among different DCIS, one of which was previously shown to recognize a protein from the Epstein-Bar virus. To elucidate mechanisms of immune evasion in IDC, we performed IF analysis of immune checkpoint proteins PD-L1 and TIGIT and found significant differences between DCIS and IDC. TIGIT-expressing T cells were more slightly frequent in DCIS than in IDC. PD-L1 expression was higher in the epithelial cancer cells in triple negative IDC compared to DCIS, and amplification of CD274 (encoding PD-L1) was only detected in triple negative IDCs. Given the close proximity of ERBB2 (encoding HER2) to a cluster of genes encoding several chemokines, we analyzed the HER2+ samples from the TCGA. We found that co-amplification of 17q12 chemokine cluster (CC) with ERBB2 was enriched in HER+ER+ luminal-like tumors but not in the HER2+ER breast tumors. We also found higher expression of both T cell activation and inhibition-related genes in tumors that lack CC gain. Also by assessing tumor samples by multicolor FISH and IF, we determined that there is an inverse correlation between CC amplification and activation of CD8+ T cells. Overall our results show co-evolution of cancer cells and the immune microenvironment during tumor progression.
Citation Format: Carlos R. Gil del Alcazar, SungJin Huh, Muhammad B. Ekram, Anne Trinh, Lin L. Liu, Francisco Beca, Zi Xiaoyuan, Misuk Kwak, Helga Bergholtz, Ying Su, Lina Ding, Lina Ding, Hege G. Russnes, Andrea L. Richardson, Kirsten Babski, Elizabeth Min Hui Kim, Charles H. McDonnell, III, Jon Wagner, Ron Rowberry, Gordon Freeman, Deborah Dillon, Therese Sorlie, Lisa M. Coussens, Judy E. Garber, Rong Fan, Kristie Bobolis, Joon Jeong, So Yeon Park, Franziska Michor, Kornelia Polyak. Immune-related changes in breast cancer tumor evolution [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A15.