Stromal B Lymphocytes Affecting Prognosis in Triple-Negative Breast Cancer by Opal/TSA Multiplexed Immunofluorescence

Min Fang,1,2 Wei Yin,3 Chunyan Qiu,4 Tao Song,2 Baihua Lin,2 Ying Wang,2 Hanchu Xiong,2 Shixiu Wu1 1Department of Radiation Oncology, The Affiliated Hangzhou Hospital of Nanjing Medical University, Hangzhou, Zhejiang, People’s Republic of China; 2Cancer Center, Department of Radiation Oncology, Zhejiang Provincial People’s Hospital(Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, People’s Republic of China; 3Department of Radiation Oncology, Hangzhou Cancer Hospital, Hangzhou, Zhejiang, People’s Republic of China; 4National Cancer Center/National Clinical Research Center for Cancer/ Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, People’s Republic of ChinaCorrespondence: Wei Yin, Department of Radiation Oncology, Hangzhou Cancer Hospital, Hangzhou, Zhejiang, People’s Republic of China, Email hzyinwei@live.cn Shixiu Wu, Department of Radiation Oncology, The Affiliated Hangzhou Hospital of Nanjing Medical University, Hangzhou, Zhejiang, People’s Republic of China, Email wushixiu@yeah.netObjective: Immune cells play a key role in tumor microenvironment. The purpose of this study was to investigate the infiltration and clinical indication of immune cells including their combined prognostic value in microenvironment of triple negative breast cancer.Methods: We investigated 100 patients with triple negative breast cancer by Opal/Tyramide Signal Amplification multispectral immunofluorescence between 2003 and 2017 at Zhejiang Provincial people’s Hospital. Intratumoral and stromal immune cells of triple negative breast cancer were classified and quantitatively analyzed. Survival outcomes were compared using the Kaplan–Meier method and further analyzed with multivariate analysis.Results: Infiltration level of stromal B lymphocytes, stromal and intratumoral CD8+ T cells, stromal CD4+ T cells, stromal PD-L1 and intratumoral tumor associated macrophages 2 cells were shown as independent factors affecting disease-free survival and overall survival in univariate analysis. Stromal B lymphocytes, T stage, N stage and pathological type were independent predictive factors for both DFS and OS in multivariate analysis. We firstly found that patients with B lymphocytes-enriched subtypes have a better prognosis than those with T lymphocytes-enriched subtypes and tumor-associated macrophage-enriched subtypes.Conclusion: The present study identified a bunch of immune targets and subtypes, which could be exploited in future combined immunotherapy/chemotherapy strategies for triple negative breast cancer patients.Keywords: triple negative breast cancer, B lymphocytes, T lymphocytes, tumor associated macrophages, multispectral immunofluorescence, prognosis