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Updated: Jan 10, 2026
Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells
Published on: May 9, 2025
Interassay Comparison With Digital Image Analysis of 4 Routine Human Epidermal Growth Factor Receptor 2 (HER2)
Maaike Anna Hempenius1, Mieke C Zwager1, Jeppe Thagaard2
1Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
Abstract:
Trastuzumab-deruxtecan, an antibody-drug conjugate targeting human epidermal growth factor receptor 2 (HER2), improves overall survival in patients with breast cancer showing low or ultralow HER2 expression. Differences in HER2 test results have been reported between various HER2 assays and between primary tumors and metastases, but an objective comparison with incorporation of the new HER2-ultralow cutoff values is needed. This study aimed to assess the performance of 4 routine clinical-grade HER2 assays across and between primary tumors and their metastases using digital image analysis. Primary tumors and metastases from 193 patients with breast cancer who participated in the IMaging PAtients for Cancer drug selecTion-Metastatic Breast Cancer trial were incorporated into 6 tissue microarrays. Samples were stained by 4 laboratories using their routine HER2 immunohistochemistry protocols: 4B5 ultraView, 4B5 OptiView, SP3, and HercepTest (DG44). HER2 scores were determined using digital image analysis. The 4 HER2 assays showed significant differences in HER2 status in both primary tumors and metastases. Eighty-five matched primary tumors and metastases were analyzed to investigate concordance in HER2 status. Although no significant differences were found in HER2 scores between primary tumors and metastases for SP3 and both 4B5 assays, DG44 showed significantly higher HER2 scores in the metastasis (P = .004). Concordance between primary tumors and metastases was highest for 4B5 ultraView (69.4%), followed by SP3 (61.2%) and 4B5 OptiView (51.8%). DG44 showed the most variability, with only 36.5% of matched samples receiving the same HER2 category. DG44 identified a significantly higher proportion of HER2-(ultra)low cases and showed the most variability in HER2 status between matched primary tumors and metastases compared with 4B5 and SP3. The choice of HER2 assay can lead to discrepancies in HER2 status assessment, which could directly influence patient eligibility for trastuzumab-deruxtecan treatment.
