Reproducibility of Equivocal HER2 In Situ Hybridization Groups 3 and 4, and Comparison With HER2 mRNA Expression: The Thin Line Between Amplified and Nonamplified Breast Cancers.

Baez-Navarro, Ximena;Riggi, Julia;Zylberberg, David;van der Made, Angelique;Van Bockstal, Mieke;et.al.
(2025) Archives of Pathology & Laboratory Medicine — Vol. 149, n° 12, p. 1093-1100 (2025)

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CONTEXT.—: Breast carcinomas (BCs) with equivocal HER2 (human epidermal growth factor receptor 2) immunohistochemistry are subjected to in situ hybridization (ISH) to assess HER2 copy numbers. Infrequently, dual-probe ISH also provides equivocal results, designated as ISH groups 2, 3, or 4. OBJECTIVE.—: To evaluate the reproducibility of HER2 ISH groups 3 and 4, and to compare the integrated immunohistochemistry/ISH HER2 result with HER2 mRNA expression. DESIGN.—: Dual-probe ISH slides of 50 BCs were re-evaluated by 2 independent observers. mRNA was extracted from microdissected tumor cells. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed for quantitative evaluation of HER2 mRNA, using the MammaTyper assay. RESULTS.—: Reproducibility of ISH groups 1 (amplified; n = 5) and 5 (nonamplified; n = 4) was good with only 1 case differently classified. Many group 4 (n = 28) and group 3 (n = 13) tumors were reclassified after recounting: of 41 patients, 9 and 18 might have been treated differently as based on assessment by observers 1 and 2, respectively. Concordance for MammaTyper HER2 status was 56% (n = 18/32) for the original report; 59% (n = 19/32) for observer 1; 72% (n = 23/32) for observer 2; and 63% (n = 20/32) for the majority opinion. CONCLUSIONS.—: The reproducibility of equivocal ISH groups is limited. Although HER2 ISH has long been considered as the gold standard to establish the HER2 status in BC, the equivocal "gray zone" between amplified and nonamplified BCs is prone to interobserver variability. Potential solutions could comprise the involvement of at least 3 different observers for assessment of equivocal ISH cases, and/or evaluation of HER2 mRNA as a more objective alternative method.
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Baez-Navarro, X., Riggi, J., Zylberberg, D., van der Made, A., Dubois, D., Vanderveken, J., Benhaddi, N., Duhoux, F., Vernaeve, H., Vassilieff, M., Berlière, M., Galant, C., van Deurzen, C. H. M., & Van Bockstal, M. (2025). Reproducibility of Equivocal HER2 In Situ Hybridization Groups 3 and 4, and Comparison With HER2 mRNA Expression: The Thin Line Between Amplified and Nonamplified Breast Cancers. Archives of Pathology & Laboratory Medicine, 149(12), 1093-1100. https://doi.org/10.5858/arpa.2024-0499-OA (Original work published 2025)