Intra-tumoral spatial heterogeneity in breast cancer quantified using high-dimensional protein multiplexing and single cell phenotyping

Abstract Background Breast cancer is a highly heterogeneous disease where variations of biomarker expression may exist between individual foci of a cancer (intra-tumoral heterogeneity). The extent of variation of biomarker expression in the cancer cells, distribution of cell types in the local tumor...

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Main Authors: Alison M. Cheung, Dan Wang, Mary Anne Quintayo, Yulia Yerofeyeva, Melanie Spears, John M. S. Bartlett, Lincoln Stein, Jane Bayani, Martin J. Yaffe
Format: Article
Language:English
Published: BMC 2025-05-01
Series:Breast Cancer Research
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Online Access:https://doi.org/10.1186/s13058-025-02038-1
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Summary:Abstract Background Breast cancer is a highly heterogeneous disease where variations of biomarker expression may exist between individual foci of a cancer (intra-tumoral heterogeneity). The extent of variation of biomarker expression in the cancer cells, distribution of cell types in the local tumor microenvironment and their spatial arrangement could impact on diagnosis, treatment planning and subsequent response to treatment. Methods Using quantitative multiplex immunofluorescence (MxIF) imaging, we assessed the level of variations in biomarker expression levels among individual cells, density of cell cluster groups and spatial arrangement of immune subsets from regions sampled from 38 multi-focal breast cancers that were processed using whole-mount histopathology techniques. Molecular profiling was conducted to determine the intrinsic molecular subtype of each analysed region. Results A subset of cancers (34.2%) showed intra-tumoral regions with more than one molecular subtype classification. High levels of intra-tumoral variations in biomarker expression levels were observed in the majority of cancers studied, particularly in Luminal A cancers. HER2 expression quantified with MxIF did not correlate well with HER2 gene expression, nor with clinical HER2 scores. Unsupervised clustering revealed the presence of various cell clusters with unique IHC4 protein co-expression patterns and the composition of these clusters were mostly similar among intra-tumoral regions. MxIF with immune markers and image patch analysis classified immune niche phenotypes and the prevalence of each phenotype in breast cancer subtypes was illustrated. Conclusions Our work illustrates the extent of spatial heterogeneity in biomarker expression and immune phenotypes, and highlights the importance of a comprehensive spatial assessment of the disease for prognosis and treatment planning.
ISSN:1465-542X