Intratumoral Estrogen Receptor Heterogeneity of Expression in Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer as Evaluated by a Brightfield Multiplex Assay.

Breast cancer (BC) is a heterogeneous disease with evolving genetic alterations and expressions of receptor proteins. Intratumoral heterogeneity (ITH) is considered to be a resistance factor in response to targeted therapies. The current single-slide, single-marker immunohistochemistry techniques cannot accurately assess ITH at the individual cancer cell level. In this study, we develop a novel brightfield multiplex assay to simultaneously assess estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) protein markers, together with the gene and the centromere of chromosome 17 () copy numbers, using a single tissue section. The data presented herein demonstrate heterogeneous cancer cell subpopulations in 11 HER2-positive/ER-positive (HER2+/ER+) tumors among 33 BCs analyzed immunohistochemically (HER2 score of 2+ or 3+). The predominant cancer cell subpopulation was HER2+/ER- (50.18%), followed by HER2+/ER+ (39.05%), HER2-/ER+ (4.26%), ER- with HER2 microheterogeneity cancer cells (3.58%), and ER+ with HER2 microheterogeneity cancer cells (2.93%). The three other tumor subtypes, namely, HER2-/ER+, HER2+/ER-, and HER2-/ER-, were more homogeneous, representing 82.59%, 99.22%, and 100% of cancer cells, respectively. This novel assay revealed that HER2+ cancer cells were more predominant than ER+ cancer cells in HER2+/ER+ tumors and provided new insights toward our understanding of BC carcinogenesis.