Multiplex immunohistochemistry and high-throughput image analysis for evaluation of spatial tumor immune cell markers in human breast cancer

Article type: Research Article

Authors: Su, Timothy^{a; 1} | Wang, Shuyang^{a; b; c; 1} | Huang, Shuya^{a; d} | Cai, Hui^a | McKinley, Eliot T.^{e; f; g} | Beeghly-Fadiel, Alicia^a | Zheng, Wei^a | Shu, Xiao-Ou^a | Cai, Qiuyin^{a; *}

Affiliations: [a] Division of Epidemiology, Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA | [b] Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, China | [c] Shanghai Key Laboratory of Medical Imaging Computing and Computer Assisted Intervention, Shanghai, China | [d] Department of Breast Surgery, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China | [e] Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, TN, USA | [f] Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA | [g] Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

Correspondence: [*] Corresponding author: Qiuyin Cai, Division of Epidemiology, Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, 1161 21st Avenue South, MCN B-2104, Nashville, TN 37232, USA. E-mail: [email protected].

Note: [1] These authors contributed equally to this work.

Abstract: BACKGROUND: The clinicopathological significance of spatial tumor-infiltrating lymphocytes (TILs) subpopulations is not well studied due to lack of high-throughput scalable methodology for studies with large human sample sizes. OBJECTIVE: Establishing a cyclic fluorescent multiplex immunohistochemistry (mIHC/IF) method coupled with computer-assisted high-throughput quantitative analysis to evaluate associations of six TIL markers (CD3, CD8, CD20, CD56, FOXP3, and PD-L1) with clinicopathological factors of breast cancer. METHODS: Our 5-plex mIHC/IF staining was shown to be reliable and highly sensitive for labeling three biomarkers per tissue section. Through repetitive cycles of 5-plex mIHC/IF staining, more than 12 biomarkers could be detected per single tissue section. Using open-source software CellProfiler, the measurement pipelines were successfully developed for high-throughput multiplex evaluation of intratumoral and stromal TILs. RESULTS: In analyses of 188 breast cancer samples from the Nashville Breast Health Study, high-grade tumors showed significantly increased intratumoral CD3+CD8+ cytotoxic T lymphocyte density (P= 0.0008, false discovery rate (FDR) adjusted P= 0.0168) and intratumoral PD-L1 expression (P= 0.0061, FDR adjusted P= 0.0602) compared with low-grade tumors. CONCLUSIONS: The high- and low-grade breast cancers exhibit differential immune responses which may have clinical significance. The multiplexed imaging quantification strategies established in this study are reliable, cost-efficient and applicable in regular laboratory settings for high-throughput tissue biomarker studies, especially retrospective and population-based studies using archived paraffin tissues.

Keywords: Multiplex immunohistochemistry, quantitative imaging analysis, breast cancer, tumor-infiltrating lymphocytes

DOI: 10.3233/CBM-220071

Journal: Cancer Biomarkers, vol. 35, no. 2, pp. 193-206, 2022