Single-cell multiplex immunocytochemistry in cell block preparations of metastatic breast cancer confirms sensitivity of GATA-binding protein 3 over gross cystic disease fluid protein 15 and mammaglobin

Q: What is the main goal of the multiplex ICC study in metastatic breast cancer cell blocks?

To validate multiplex immunocytochemistry (mICC) for GATA3, GCDFP15, and MMG in cell block preparations and to use single-cell expression patterns to identify the most sensitive single markers and marker combinations for different breast cancer subtypes.

Q: How were GATA3, GCDFP15, and MMG multiplexed for analysis?

They were paired with specific chromogens (OptiView 3 with DAB for GATA3; Ventana DISCOVERY Purple and Blue for GCDFP15 and MMG) along with cyclical and serial staining, followed by bright-field imaging and digital analysis.

Q: Which marker showed the highest sensitivity when used as a single marker?

GATA3 displayed the highest sensitivity as a single marker across breast cancer subtypes, with combination effects varying by subtype.

Sep 21, 2026

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Metastatic breast cancers frequently require immunocytochemistry (ICC) in cytology specimens. This study validates multiplex ICC (mICC) for GATA-binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP15), and mammaglobin (MMG) in cell block preparations, using single-cell expression pattern analysis to determine the most sensitive markers and marker combinations across breast cancer subtypes. Traditional and mICC are compared with single-cell phenotyping supported by imaging analysis and pathologist confirmation.

LanguageEnglish

CategoryResearch & Report

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Abstract

Background

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Materials and Methods

DOI: 10. 1002/cncy.22910 ORIGINAL ARTICLE Single‐cell multiplex immunocytochemistry in cell block preparations of metastatic breast cancer confirms sensitivity of GATA‐binding protein 3 over gross cystic disease fluid protein 15 and mammaglobin Joshua J. X. Li MBChB1 | Hiu Yu Cheng MSc2 | Conrad H. C. Lee MSc2 | Joanna K. M. Ng MBBS2,3 | Julia Y. Tsang PhD2 | Gary M. Tse MBBS2,3 1Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong 2Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, Hong Kong 3Department of Pathology, North District Hospital, Sheung Shui, New Territories, Hong Kong Correspondence Gary M. Tse, Department of Anatomical and Cellular Pathology, 1/F, Clinical Sciences Building, Prince of Wales Hospital, Ngan Shing St, Shatin, Hong Kong. Email: [garytse@cuhk.edu.hk](garytse@cuhk.edu.hk) Abstract Background: Metastatic breast cancers are frequently encountered in cytology and require immunocytochemistry (ICC). In this study, traditional and multiplex ICC (mICC) for GATA‐binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP15), and mammaglobin (MMG) were performed with the aim of validating mICC in cell blocks, with further single‐cell expression pattern analysis to identify the single markers and combinations of markers most sensitive in subtypes of breast cancer. Methods: GATA3, GCDFP15, and MMG were paired with OptiView 3,30 ‐diaminobenzidine and Ventana DISCOVERY Purple and Blue, respectively, with cyclical and serial staining. Bright‐field imaging was performed with the Mantra 2 system and analyzed with the inForm Tissue Finder (Akoya Biosciences). Cell detection and phenotyping were further confirmed by two pathologists. Results: In the 36 cases studied, traditional ICC and mICC demonstrated good concordance (kappa coefficient, >0. 5; p < .01) at three cutoffs (1%, 5%, and 50%), except for GATA3 at the 1% cutoff. Single‐marker positivity outnumbered double‐ marker positivity and the exceedingly rare triple‐marker positivity (<3%). GATA3 was the leading single marker–positive phenotype in all breast cancer subtypes, except for MMG in estrogen receptor–positive, progesterone receptor–positive, and human epidermal growth factor receptor 2–positive (ERþ/PRþ/HER2þ) breast cancers. Limited to two markers, GATA3/MMG included the greatest number of tumor cells for luminal breast cancers (ERþ/PRþ/HER2þ, 60. 6%; ERþ/PRþ/ HER2þ, 31.4%), whereas HER2‐overexpressed breast cancers (27.4%) and triple‐ negative breast cancers (26.4%) favored the combination of GATA3/GCDFP15. Conclusions: For a single marker, GATA3 displayed the highest sensitivity. The addition of MMG for hormone receptor‐positive breast cancers and GCDFP15 for This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2024 The Author(s). Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society. Cancer Cytopathol. 2025;e22910. wi[leyonlinelibrary.com/journal/cncy](leyonlinelibrary.com/journal/cncy- 1 of 9)[-](leyonlinelibrary.com/journal/cncy- 1 of 9)[ 1 of 9](leyonlinelibrary.com/journal/cncy- 1 of 9) [https://doi.org/10.1002/cncy.22910](https://doi.org/10.1002/cncy.22910) hormone receptor‐negative breast cancers further increased sensitivity. The low proportion of multimarker‐positive cells suggested that the coexpression observed with traditional ICC is attributable to intratumoral heterogeneity, not genuine coexpression. KEYWORDS breast cancer, cell block, immunocytochemistry, multiplex INTRODUCTION Metastatic breast cancers are frequently encountered in routine cytology practice. 1,2 GATA‐binding protein 3 (GATA3), gross cys

FAQs

What is the main goal of the multiplex ICC study in metastatic breast cancer cell blocks?

To validate multiplex immunocytochemistry (mICC) for GATA3, GCDFP15, and MMG in cell block preparations and to use single-cell expression patterns to identify the most sensitive single markers and marker combinations for different breast cancer subtypes.

How were GATA3, GCDFP15, and MMG multiplexed for analysis?

They were paired with specific chromogens (OptiView 3 with DAB for GATA3; Ventana DISCOVERY Purple and Blue for GCDFP15 and MMG) along with cyclical and serial staining, followed by bright-field imaging and digital analysis.

Which marker showed the highest sensitivity when used as a single marker?

GATA3 displayed the highest sensitivity as a single marker across breast cancer subtypes, with combination effects varying by subtype.

Sep 21, 2026

Uploaded by Aurelia