Abstract P4-04-09: Systematic analysis of immune cell composition revealed immunological profile of breast cancer microenvironment represented by histologically assessed tumor-infiltrating lymphocyte and PD-L1 expression
Authors: Hanamura Toru , Kitano Shigehisa , Kagamu Hiroshi , Yamashita Makiko , Terao Mayako , Tsuda Banri , Okamura Takuho , Kumaki Nobue , Hozumi Katsuto , Harada Naoki , Iwamoto Takaiki , Honda Chikako , Kurozumi Sasagu , Niikura Naoki 
Published: March 2022

Abstract
Purpose. The clinical use of immune checkpoint inhibitors for multiple cancers has attracted attention in tumor immunology. Emerging evidence suggests that a better understanding of tumor immunology will lead to the development of new treatment strategies or the effective use of existing therapies. Histologically assessed tumor-infiltrating lymphocytes (hTIL) and programmed cell death 1 ligand 1 (hPD-L1) have been established as prognostic or predictive biomarkers in certain subsets of breast cancer. In addition, researchers have shifted their focus to the various immune cell subsets that make up TILs. However, the complexity of multiple types of immune cells in TIL or PD-L1 expressing cells is not fully understood. In this analysis, the immune cell fraction in breast cancer tissue and blood was evaluated by multicolor flow cytometry (FCM) to analyze the association between them and hTIL and hPD-L1. Methods. Forty-five tumor and 18 blood samples were collected from breast cancer patients. The leukocyte count, proportion of 11 types of immune cell fraction, and PD-L1 expression of each fraction were evaluated by FCM for both tumor and blood samples. The immune cell fractions are classified into the following categories based on the expression of cell surface markers: leukocyte, total T cell (total T), CD4+ T cell (CD4+ T), CD8+ T cell (CD8+T), B cell (B), monocyte/macrophage (Mo/Mϕ), nonclassical monocyte (CD16+Mo), myeloid-derived suppressor cells (MDSC), dendritic cells (DC), myeloid dendritic cells (mDC), natural killer cells (NK), minor NK, and natural killer T cells (NKT). hTIL, and hPD-L1 were evaluated by H-E staining and immunohistochemistry, respectively. Results. The mean density and interquartile range of tumor-infiltrating leukocytes were similar to those in previous report. For the immune cell fraction in the leukocytes of tumor tissue, the main population consisted of CD8+T and CD4+T, which showed a similar trend to that of blood. The proportions of DC, mDC, NK, and minor NK in tumor tissues were positively correlated with those of blood. When the percentage of each immune cell fraction of tumor tissue and that of blood were compared, the proportions of DC, mDC, and minor NK were significantly higher in tumor tissues than those in blood samples, and the proportions of CD4+T and NK were significantly lower in tumor tissue than in blood. No significant association was found between blood immune cell composition and hTIL or hPD-L1. High hTIL levels were associated with high leukocyte infiltration, high proportions of CD4+ T and CD8+ T, and a low proportion of NK and NKT in the tumor tissue. When PD-L1 positive cell percentage of each immune cell fraction was compared between the tumor tissue and blood, PD-L1 positive ratios were significantly higher in tumor tissue than in blood for all lineages except for lymphoid fractions. For tumor tissues, PD-L1 expression was high in Mo/Mϕ, CD16+Mo, MDSC, DC, and mDC. hPD-L1 positivity was associated with PD-L1 expression in Mo/Mϕ, CD16+Mo, DC, and mDC. Conclusion. Comprehensive analysis of the immune cell fractions revealed the immunological profile of breast cancer tissue represented by hTIL or hPD-L1. Our data indicate that hTIL not only reflects the amount of immune cell infiltration but also reflects a state in which acquired immunity is activated relative to innate immunity. Non-B cell antigen-presenting cell fractions such as Mo/Mϕ, CD16+ Mo, MDSC, DC, and mDC were primarily involved in the PD-L1 pathway in breast cancer microenvironments. In addition, hPD-L1 reflects PD-L1 expression in these immune cell fractions. Our data provide a basic understanding of the immune response in the breast cancer microenvironment and contribute to further development of tumor immunology.
Citation Format: Toru Hanamura, Shigehisa Kitano, Hiroshi Kagamu, Makiko Yamashita, Mayako Terao, Banri Tsuda, Takuho Okamura, Nobue Kumaki, Katsuto Hozumi, Naoki Harada, Takaiki Iwamoto, Chikako Honda, Sasagu Kurozumi, Naoki Niikura. Systematic analysis of immune cell composition revealed immunological profile of breast cancer microenvironment represented by histologically assessed tumor-infiltrating lymphocyte and PD-L1 expression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-09.