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. 2025 Nov;29(6):849-857.
doi: 10.1007/s40291-025-00814-5. Epub 2025 Sep 6.

Inter-Assay Variability of TROP2 Immunohistochemistry in Triple-Negative Breast Cancer

Giulia Cursano #  1   2 , Alberto Concardi #  1   2 , Mariia Ivanova  1 , Chiara Frascarelli  1   2 , Eltjona Mane  1 , Elisa Mangione  1   2 , Stefano Santaguida  2   3 , Daniela Tosoni  3 , Salvatore Pece  2   3 , Antonio Marra  4 , Carmen Criscitiello  2   4 , Giuseppe Curigliano  2   4 , Giuseppe Viale  1   2 , Konstantinos Venetis  5 , Elena Guerini Rocco #  1   2 , Nicola Fusco #  1   2
Affiliations

Inter-Assay Variability of TROP2 Immunohistochemistry in Triple-Negative Breast Cancer

Giulia Cursano et al. Mol Diagn Ther. 2025 Nov.

Abstract

Background and objective: Sacituzumab govitecan, an anti-trophoblast cell surface antigen 2 (TROP2) antibody-drug conjugate, has been approved by both the US Food and Drug Administration and European Medicines Agency for patients with metastatic triple-negative breast cancer who have received two or more prior systemic therapies, including at least one of them for advanced disease. Although TROP2 evaluation is not required for patient selection, survival data from the ASCENT trial show improved response rates in patients with high TROP2 expression by immunohistochemistry. However, there is no standardized testing assay for these patients. This study evaluated the consistency of TROP2 expression analysis across different immunohistochemistry assays.

Methods: Twenty-six triple-negative breast cancer samples were analyzed using three different immunohistochemistry assays on a Dako Omnis platform, according to manufacturer protocols. Specifically, ENZO-ABS380-0100 (assay A, used in ASCENT), Abcam SP295 (assay B, used in TROPiCS-02), and Santa Cruz B9-sc-376746 (assay C, used in cross-sectional studies). TROP2 expression on tumor cell membranes was quantified using the H-score, categorized as low (≤ 100), intermediate (> 101 to ≤ 200), and high (> 200). Assay agreement was evaluated using Cohen's κ and Gwet's AC2 statistics.

Results: Assay A showed a broader range of TROP2 expression, with 57.7% of samples (n = 15) classified as low, 34.6% (n = 9) as intermediate, and 7.7% (n = 2) as high expressors. Assay B identified only n = 5 (19.2%) low expressors, n = 11 (42.3%) intermediate, and n = 10 (38.4%) high. While assay C identified n = 4 (15.4%) low expressors, n = 12 (46.2%) intermediate, and n = 10 (38.4%) high. Not surprisingly, assays B and C exhibited substantial agreement, with 80.8% of cases showing consistent results (κ = 0.81; p < 0.0001), indicating similar staining outcomes for TROP2 expression. The overall concordance between Assay A, B, and C was fair to moderate (AC2 = 0.35, p = 0.0067).

Conclusions: Our hypothesis-generating study highlights significant variability among TROP2 assays, suggesting differences in sensitivity and specificity for triple-negative breast cancer. We demonstrate that TROP2 expression is both heterogeneous and dynamic across samples and assays, highlighting the need for methodological improvements in testing. Future research integrating computational pathology with standardized immunohistochemistry protocols and quantitative scoring systems may enhance the clinical utility of TROP2 as a biomarker in triple-negative breast cancer.

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Conflict of interest statement

Declarations. Conflicts of Interest/Competing Interests: Giulia Cursa from Veracyte. Antonio Marra has received support from Menarini Group and served on the speakers’ bureau for Roche and AstraZeneca. Carmen Criscitiello has participated in advisory or consultancy roles and speakers’ bureau engagements for Eli Lilly, Pfizer, Novartis, Roche, AstraZeneca, MSD, Daiichi Sankyo, Gilead, and Seagen. Giuseppe Curi has received honoraria for speaker engagements from Roche, Seattle Genetics, Novartis, Lilly, Pfizer, Foundation Medicine, NanoString, Samsung, Celltrion, BMS, and MSD; honoraria for consultancy from Roche, Seattle Genetics, and NanoString; honoraria for participation in advisory boards from Roche, Lilly, Pfizer, Foundation Medicine, Samsung, Celltrion, and Mylan; honoraria for writing engagements from Novartis and BMS; and honoraria for participation in the Ellipsis Scientific Affairs Group. He has also received institutional research funding for conducting phase I and II clinical trials from Pfizer, Roche, Novartis, Sanofi, Celgene, Servier, Orion, AstraZeneca, Seattle Genetics, AbbVie, Tesaro, BMS, Merck Serono, Merck Sharp & Dohme, Janssen-Cilag, Philogen, Bayer, Medivation, and Medimmune. Konstantinos Venetis has received honoraria for speakers’ bureau participation from Merck Sharp & Dohme (MSD), Roche, and AstraZeneca. Elena Guerini Rocco has received advisory fees, honoraria, travel accommodations/expenses, grants, and/or non-financial support from AstraZeneca, Exact Sciences, GSK, Illumina, MSD, Novartis, Roche, and Thermo Fisher Scientific. Nicola Fusco has received honoraria for consulting, advisory roles, speakers’ bureau participation, travel, and/or research grants from Merck Sharp & Dohme (MSD), Merck, Novartis, AstraZeneca, Roche, Menarini Group, Daiichi Sankyo, GlaxoSmithKline (GSK), Gilead, Sysmex, Veracyte Inc., Sakura, Leica Biosystems, Lilly, Pfizer, and AbbVie. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and/or in the decision to publish the results. Alberto Concardi, Mariia Ivanova, Chiara Frascarelli, Eltjona Mane, Elisa Mangione, Stefano Santaguida, Daniela Tosoni, Salvatore Pece, Antonio Marra, and Giuseppe Viale have no conflicts of interest that are directly relevant to the content of this article. Ethics Approval: This study was conducted in accordance with ethical guidelines and approved by the local ethics committee (approval number #UID3472). Informed consent was obtained from all patients for the use of tissue samples. All patient information was pseudoanonymized in compliance with the European Union’s General Data Protection Regulation. Consent to Participate: Not applicable. Consent for Publication: Not applicable. Availability of Data and Material: All data generated or analyzed during this study are included in this published article. Code Availability: Not applicable. Authors’ Contributions: GC: writing (original draft), methodology. AC: writing (original draft), methodology. MI: writing (original draft), software, methodology. CF: writing (review and editing), methodology, statistical analyses. Eltjona Mane: writing (review and editing), methodology. Elisa Mangione: writing (review and editing). SS: writing (review and editing). DT: writing (review and editing). SP: writing (review and editing). AM: writing (review and editing). CC: writing (review and editing). GC: writing (review and editing). KV: writing (review and editing), project administration. EG-R: writing (review and editing), methodology. NF: writing (review and editing), project administration, conceptualization.

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