Not All HER2-Positive Breast Cancers Are the Same: Intratumoral Heterogeneity, Low-Level HER2 Amplification, and Their Impact on Neoadjuvant Therapy Response

Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are frequently treated with neoadjuvant anti-HER2 and chemotherapy (NACT). However, treatment response varies, with a subset of tumors showing high residual cancer burden (RCB). This study investigates the relationship between HER2 immunohistochemical (IHC) intratumoral heterogeneity (ITH), low-level HER2 amplification, and tumor response to NACT. A total of 205 post-NACT HER2-positive breast carcinomas with available RCB results were classified into the following 5 HER2 groups: (1) IHC 3+ (HER2 IHC positive, no fluorescence in situ hybridization performed), (2) Group 1-High (fluorescence in situ hybridization HER2 copies > 8 or HER2/CEP17 ratio > 4), (3) Group 1-Intermediate (HER2 copies > 6-8 or HER2/CEP17 ratio > 3-4), (4) Group 1-Low (HER2 copies 4-6 and HER2/CEP17 ratio 2-3), and (5) Group 3 (HER2 copies ≥ 6 and HER2/CEP17 ratio < 2). Low-level HER2 amplification, collectively designated as HER2 copies 4 to 8 or HER2/CEP17 ratio < 4, was associated with reduced response to HER2-targeted therapy and higher RCB post-NACT. HER2 IHC ITH, defined as the presence of at least 3 distinct staining intensities, with at least 10% of tumor cells exhibiting weak or no staining, was significantly more prevalent in low-level HER2 amplification groups (Group 1-Intermediate: 93.3%, Group 1-Low: 87.5%, and Group 3: 80.0%) compared with high-level amplification groups (IHC 3+: 24.7% and Group 1-High: 28.6%) (P < .001). Both low-level HER2 amplification and HER2 IHC ITH, regardless of hormone receptor status, were independently associated with poor treatment response, and tumors demonstrating both features had the highest likelihood of low therapeutic efficiency. These findings suggest that both low-level HER2 amplification and HER2 IHC ITH contribute to poor NACT response and may warrant alternative therapeutic strategies. Further prospective studies are needed to refine the clinical significance of low-level HER2 amplification and IHC ITH, particularly in the context of novel HER2-targeted therapies such as antibody-drug conjugates.