Qualification of a multiplexed tissue imaging assay and detection of novel patterns of HER2 heterogeneity in breast cancer

Key findings

• Qualified a breast-cancer CyCIF antibody panel against clinical standards. HER2, ER, and PR fluorophore-conjugated antibodies were benchmarked against clinical IHC and HER2 FISH across 866 tissue cores from 294 patients, using pixel-, cell-, and tissue-core-level analyses to confirm concordance with the established clinical antibodies.
• Single-cell imaging resolved four HER2-heterogeneous cell clusters. Analysis of 589,343 cells from 278 breast carcinomas — 567 HER2-positive tumors from 189 patients — identified four distinct clusters with heterogeneous HER2 expression that also varied in ER, PR, p53, AR, and PD-L1.
• The qualified panel was expanded to profile the tumor and its immune context. The three clinically benchmarked antibodies were combined with seven additional markers (CD45, CD68, PD-L1, p53, Ki67, pRB, AR), and heterogeneity scores derived from the four clusters showed correlations to HER2 intratumoral heterogeneity with potential relevance to clinical outcomes.

CyteFinder in the methods

“CyCIF ( https://www.cycif.org/ ) was performed as described previously 37 and used by our group 37 , 55 , 56 . Briefly, 4–5 µm FFPE unstained slides were baked (30 mins at 60 °C) and antigen retrieval was performed using Leica BOND RX with ER1 solution (Leica Biosystems #AR9961). A pre-staining cycle is subsequently performed and is constituted by blocking of sample with secondary antibodies so that auto-fluorescence and non-specific antibody binding can be reduced. All staining steps were done at 4 °C overnight. Staining is followed by bleaching with 25 mM NaOH with 4.5% H 2 O 2 for 45 mins with light exposure. Each successive CyCIF cycle included immunostaining the specimen with the testing antibodies, followed by nuclear staining with a DNA dye, four-channel imaging and fluorophore bleaching. When all cycles are completed, the slide is stained with H&E to allow conventional histopathology review. Individual images are then stitched together into high-dimensional representation for further segmentation and analyses. The RareCyte CyteFinder (RareCyte, Seattle, WA) was used for image capturing.”

— Guerriero et al., npj Breast Cancer (2024), Methods, “Tissue-based cyclic immunofluorescence”

Disclosure: RareCyte is named in the competing-interests statement of the publication cited above.

Why it matters for CyteFinder users

If you are weighing CyteFinder for tissue imaging, look at the specific job it did here. The question was whether a research multiplex assay could reproduce what clinical HER2 IHC and FISH already report, and then go further by resolving how HER2 expression varies from cell to cell. The CyteFinder was the acquisition instrument: it captured the four-channel fluorescence images for every CyCIF cycle across 866 tissue cores, producing the whole-slide, single-cell image data the authors then registered, segmented, and quantified. The staining chemistry and image stitching came from separate tools; the CyteFinder's contribution was the imaging that made a single-cell readout possible. Two things follow for your own work. Because the same section is imaged over successive cycles, you can benchmark a panel against the clinical standards your pathologists already trust, at pixel, cell, and core level. And because the readout is single-cell, you can measure heterogeneity directly, showing four distinct HER2 clusters within one tumor rather than a single averaged score. That distinction is what let this study move from panel qualification to a new description of HER2 heterogeneity.