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.

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 3,3'-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 (ER+/PR+/HER2+) breast cancers. Limited to two markers, GATA3/MMG included the greatest number of tumor cells for luminal breast cancers (ER+/PR+/HER2+, 60.6%; ER+/PR+/HER2+, 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 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.