Exploring the Heterogeneity of HER2 gene status and Expression in Non-Positive Breast Cancer Patients: Insights from Immunohistochemistry and Fluorescence In Situ Hybridization

Breast cancer became the most prevalent malignancy among women, and HER2 expression status is critical for treatment decisions. With the emergence of ADC drugs, HER2 low-expressing patients who previously did not respond well to traditional anti-HER2 therapies may now benefit. In this study, we evaluated HER2 expression in 349 HER2-non-positive breast cancer patients using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Our analysis revealed that HER2-low tumors exhibited fewer grade III tumors (39.74% and 55.65%, respectively, P = 0.005) and higher positivity for estrogen receptor (ER, 88.89% vs. 61.74%, P < 0.001) and progesterone receptor (PR, 84.62% vs. 57.39%, P < 0.001) compared to HER2-0 tumors. IHC interpretation varied widely, with antibodies showing only 67.3% and 65.3% agreement in HER2-0 and HER2-2 + groups (P < 0.001), respectively. Similarly, pathologists showed limited agreement, with 66.4% and 63.7% consistency in HER2-0 and HER2-2 + groups (P < 0.001). FISH analysis revealed significant differences in HER2 gene signals between HER2-0 and HER2-low tumors, but no clear cut-off value could be identified. Notably, HER2 gene red signal averages were mostly ≥ 2 and < 4, with HER2-0 tumors primarily ≤ 2.5, and HER2/CEP17 ratio mostly between 1 and 2, with HER2-0 tumors primarily ≤ 1.4. Despite distinct clinicopathological features, FISH remains inadequate for distinguishing HER2-low from HER2-0 expression. Future studies are needed to improve HER2 assessment in this challenging subset of patients.