Immunohistochemical (IHC) evaluation has shown that human epidermal growth factor receptor 2 (HER2) may not be expressed homogeneously among all cancer cells within a given tumor. The clinical significance of intratumoral HER2 heterogeneity is unclear. Exploration of tumor heterogeneity is facilitated by tissue imaging technologies such as t-CyCIF, a highly multiplexed immunofluorescence microscopy technique that permits visualization of up to 60 antigens and analysis on a single cell level from formalin-fixed, paraffin-embedded tissue. To utilize t-CyCIF for the evaluation of breast tumors, this study was undertaken to: 1) validate antibodies to be used against the clinically relevant markers HER2, estrogen receptor (ER) and progesterone receptor (PR), and 2) use these antibodies along with other validated antibodies to define the tumor microenvironment (TME) to interrogate breast tumors at a single cell level.


T-CyCIF is an iterative whole-slide imaging process, in which successive four-channel images, each involving different antibodies, are collected from the same sample and then merged to generate a high-dimensional representation used for visualization and analysis. In phase one of this study, 948 tissue cores (representing 295 patients in triplicate) were used to validate HER2, ER, and PR antibodies against a single antibody commonly used in clinical practice as a reference. Analyses were performed at the level of tissue cores, cells and pixels. Inter-assay analyses were performed comparing: t-CyCIF vs. IHC, the latter assessed by digital pathology and two pathologists; and also, t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. In the second phase, following selection of validated HER2, ER and PR antibodies, expression of CD45, CD68, PD-L1, p53, Ki67, pRB and the androgen receptor (AR) were evaluated at a single cell level in 312 HER2+ invasive breast cancer samples, representing 104 patients, to better understand the TME, cancer cell heterogeneity and the cell identities/states present in breast carcinomas.


In the first phase of the study, 13 different ER, PR or HER2 antibodies were analyzed. The pixel-to-pixel evaluation, which evaluates concordance in staining, resulted in r scores of 0.86 (ER; Pearson correlation), 0.93 (PR) and 0.94 (HER2) and correlation scores in single-cell comparisons ranged from 0.76 to 0.81. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.87 and 0.91 for ER and HER2, respectively, on t-CyCIF vs. Aperio; and 0.85 to 0.94 by pathology review) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). This resulted in validated fluorophore-conjugated antibody panels for use in t-CyCIF that correspond well to established standards. In the second phase, single cell analysis of HER2+ breast cancer was performed. Cancer cells were defined as keratin positive and using t-Distributed Stochastic Neighbor Embedding (t-SNE) seven cancer cell clusters were identified including two HER2hi clusters differing in ER, p53, AR and PD-L1 expression, two HER2lo clusters differing in PR, Ki67, pRB, p53 and AR and three HER2neg clusters differing in PR, Ki67, ER, PD-L1 and AR. Heterogeneity scores were calculated based on diversity among clusters.


This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. Using the validated antibody panel, we uncovered patterns of expression of markers relevant to breast cancer biology that correlate with HER2 high, low and negative states. Ongoing studies are looking at correlations between HER2 heterogeneity, responses to therapy and clinical outcomes.

Citation Format: Jennifer L Guerriero, Jia-Ren Lin, Ricardo G Pastorello, Ziming Du, Shaolin Mei, Krishan Taneja, Stuart J Schnitt, Deborah A Dillon, Peter K Sorger, Sandro Santagata, Elizabeth A Mittendorf. Highly multiplexed tissue-based cyclic immunofluorescence (t-CyCIF) for precision oncology identifies novel patterns of HER2 heterogeneity in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-02.