Evaluation of HER2 immunohistochemistry-positive and immunohistochemistry-negative FISH amplification breast cancers using next-generation sequencing

Objectives: In this study, we aimed to assess the concordance between HER2 positivity, defined as scores of 2+ or 3+ by immunohistochemistry (IHC), and HER2 gene amplification results obtained by fluorescence in situ hybridization (FISH) and gene panel testing (GPT). Methods: A total of 45 breast cancer patients with sufficient biopsy, breast-conserving surgery, or mastectomy specimens collected between 2018 and 2024 at Hokuto Hospital were included in the study. HER2 status was defined as HER2+ in cases with scores of 3+ on IHC staining, HER2-FISHamp in cases with scores of 2+ on IHC staining plus FISH amplification. FISH amplification was defined as a ratio of HER2 to the amplification of the chromosome 17 centromere ≥2.0. Next-generation sequencing (NGS) for these genes was conducted using MiSeq. The criterion for ERBB2 amplification requires gene amplification of more than 2 standard deviations (SD) above the average of the panel genes and CN ≥4. Results: HER2+ correlated with the presence of NGS amplification of ERBB2 (p<0.001). NGS amplification was significantly more detectable in HER2+ than in HER2-FISHamp. HER2-FISHamp correlated with larger tumor size (p=0.009) and the presence of lymph node metastases (p<0.001). No correlations were observed between the HER2 status and other parameters such as age, histological grade, stage histological type, recurrence, treatment response evaluation, and breast cancer mortality. Conclusions: To achieve a comprehensive diagnosis, it is crucial to integrate GPT findings with other diagnostic approaches and to have a though understanding of the characteristics and limitations of each method.