Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes

• Published in: Breast cancer research : BCR Vol. 23; no. 1; p. 114
• Main Authors: Cheung, Alison Min-Yan, Wang, Dan, Liu, Kela, Hope, Tyna, Murray, Mayan, Ginty, Fiona, Nofech-Mozes, Sharon, Martel, Anne Louise, Yaffe, Martin Joel
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 18.12.2021; BioMed Central; BMC
• Subjects: Annotations; Antibodies; Biomarkers; Biomarkers, Tumor - metabolism; Breast cancer; Breast cancer subtypes; Breast Neoplasms - pathology; CD20 antigen; CD8 antigen; Cyclin-dependent kinase inhibitor p21; Cyclin-dependent kinases; Diagnosis; Epidermal growth factor; ErbB-2 protein; Estrogen; Estrogen receptors; Estrogens; Female; Fluorescent Antibody Technique; Heterogeneity; Humans; Image processing; Immunofluorescence; Immunofluorescence imaging; Immunohistochemistry; INK4a protein; Kinases; Localization; Morphology; p16 Protein; p53 Protein; Progesterone; Protein multiplexing; Proteins; Receptor, ErbB-2 - metabolism; Receptors, Progesterone - metabolism; Single-Cell Analysis; Spatial arrangement; Spatial discrimination; Spatial heterogeneity; Staining and Labeling; Triple Negative Breast Neoplasms; Tumor Microenvironment; Tumor proteins; Tumors; United States > US; Heterogeneity; Immunofluorescence imaging; Protein multiplexing; Single cell imaging; Breast cancer subtypes; Biomarkers; Spatial arrangement
• ISSN: 1465-542X; 1465-5411; 1465-542X
• DOI: 10.1186/s13058-021-01475-y

The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification. Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures. On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.