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

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.