Currently, therapy for early-stage human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) is based on the combination of trastuzumab and pertuzumab plus chemotherapy in a neoadjuvant regimen. The INMUNOHER study aimed to detect immunological markers in peripheral blood and their association with treatment response. Sixty-two HER2+ BC patients were recruited. Pre-treatment samples were obtained before the start of treatment, while post-treatment samples were obtained after completing therapy and before surgery and were analyzed by flow cytometry. The pathologic complete response (pCR) rate achieved was 82.3%. The expression of the NKp30, PD-1, and TIM-3 receptors was reduced in the Natural Killer (NK)-CD56dim subset of patients who did not achieve pCR. Following therapy, many changes were found in leukocytes, including alterations in T cell lymphocyte proportions. Also, the percentage of NK cells decreased, and several phenotypic changes were observed in this population. After treatment, IFN-γ production by NK cells against HER2+-cells with or without trastuzumab was significantly reduced. HER2-targeted therapy plus chemotherapy demonstrated high efficacy in most patients, reducing the statistical power for finding immunological markers. However, NK subset phenotypes correlated better with response groups, and numerous changes in the percentage of leukocytes and T and NK cells, as well as changes in the functionality of NK cells, were observed in most patients after treatment, encouraging further research into these immune populations.

Introduction: Adaptive Natural Killer cells are a specialized NK-cell subset with enhanced antibody-dependent effector functions, making them attractive candidates for cancer immunotherapy. Although circulating adaptive NK cells have demonstrated potent antitumor activity, their presence and functional status within the breast tumor microenvironment (TME) remain largely unknown. Methods: Here, we investigated the infiltration, phenotype, functional properties, and clinical relevance of NKG2C ⁺ NK cells with adaptive features (aNK) in treatment-naïve breast cancer. Using paired peripheral blood, tumor, and adjacent non-tumoral breast tissue samples, together with functional assays and transcriptomic analyses from The Cancer Genome Atlas (TCGA), we characterized tumor-infiltrating aNK cells and evaluated their association with clinical outcome. Results: aNK cells are present within breast tumors while preserving their phenotype and enhanced effector functions. Tumor-infiltrating aNK cells retained robust antibody-dependent degranulation and IFN-γ production. Complementary studies using peripheral blood-derived aNK cells further demonstrated their superior capacity to promote dendritic cell maturation, supporting their immunoregulatory potential. Moreover, we analyzed an adaptive NK-associated transcriptional signature in TCGA breast cancer samples and found that higher signature scores were associated with a longer progression-free interval and an immune microenvironment enriched in specific CD8 ⁺ T-cell populations. Discussion: These findings indicate that aNK cells are functionally resilient to the suppressive breast TME, maintaining some specialized functions and immunoregulatory properties after tumor infiltration. Their persistence and association with favorable clinical outcome provide a rationale for further investigating their potential role in antibody-based immunotherapy and as a possible target for therapeutic strategies in BC.