Immune Profiling the Axilla with Fine Needle Aspiration Is Feasible to Risk-Stratify Breast Cancer

<b>Background</b>: Axillary lymph node (ALN) metastasis is a critical prognostic determinant in breast cancer (BC) that informs surgical management. However, surgically clearing the axilla carries morbidity, so less invasive methods of risk-stratifying patients are needed. ALN fine needle aspiration (FNA) is currently used to detect BC metastases, but these samples also contain immune cells. <b>Methods</b>: Cells obtained via FNA from BC-patient-derived ALNs were analysed using flow cytometry. <b>Results</b>: FNA acquires sufficient leukocytes for comprehensive immunophenotyping of reactive, patient-derived ALNs. All CD4⁺ and CD8⁺ T-cell subsets (naïve, terminal effector, central memory, and effector memory) and rarer (<2%) natural killer (NK) and plasmacytoid dendritic cell (pDC) populations are represented. Importantly, the immune-cell profile of one reactive ALN appears to reflect the immune status of the patient's axilla. Furthermore, FNA captures immune differences between patients with ≤1 or ≥2 metastatic ALNs. Increased numbers of naïve CD4⁺ T cells, but fewer terminal effector, central memory, and effector memory subpopulations, were obtained from patients with ≥2 metastatic ALNs. Moreover, despite their sparse distribution pattern on whole-section immunohistochemistry (WSI), FNA revealed that CD56⁺ NK cell activation receptors were decreased in patients with ≥2 metastatic ALNs. Finally, FNA captured a decrease in pDCs in patients with ≤1 metastatic ALNs, despite their clustered distribution pattern on WSI. <b>Conclusions</b>: FNA is not only feasible for sampling leukocytes from reactive, patient-derived ALNs, but also identifies immune-cell profiles that reflect axillary tumour burden in BC. Thus, this technique could be used to risk-stratify BC patients in the future.