Tertiary lymphoid structures are associated with lower axillary residual nodal burden in breast cancer patients after neoadjuvant chemotherapy.

backgroundTertiary lymphoid structures (TLSs) are associated with an improved chemotherapy response and favorable clinical outcomes in various tumor types, including breast cancer. The number of TLSs increases in breast cancer after neoadjuvant chemotherapy (NAC), potentially activating the immune landscape of the tumor microenvironment. However, the potential relationship between TLSs and clinicopathological characteristics in patients with breast cancer after receiving NAC is poorly understood.

methodsThis retrospective study included clinicopathological data from breast cancer patients treated at Sun Yat-Sen Memorial Hospital between 2012 and 2023. Hematoxylin‒eosin staining and immunohistochemical analysis were performed to evaluate TLSs and lymphatic vessels in two cohorts comprising 326 NAC-treated and 304 treatment-naive breast cancer patients, with validation conducted in a cohort of 23 paired biopsy specimens collected before and after receiving NAC. RNA fluorescence in situ hybridization (RNA-FISH) was employed to quantify lymphatic endothelial cell patterns. TLS gene expression scores were determined to validate the observed differences in TLS patterns in breast cancer patients.

resultsTLSs were more common in breast cancer samples after receiving NAC (p < 0.0001). Compared with breast cancer patients with a low density of TLSs (TLS-low) (n = 92) and with the absence of TLSs (TLS-absent) (n = 142), patients with a high density of TLSs (TLS-high) (n = 92) exhibited significantly lower stages of residual axillary disease (Bonferroni-adjusted p < 0.0167) after receiving NAC, but there were no clinical stage differences before receiving NAC (p > 0.05). Compared with patients with a low density of TLSs after receiving NAC, patients with a high density of TLSs exhibited a lower residual axillary nodal burden (p < 0.0001). Transcriptomic analysis validated that patients with NAC-treated breast cancer with an axillary pathological complete response (pCR) attained higher TLS gene scores than those with a non-pCR axilla did (p < 0.0001). Lymphatic vessel density and size were highly correlated with TLS density in the NAC cohort, and these results agree with those of a validation cohort (n = 23) that included a paired-sample set of breast cancer patients before and after receiving NAC. The RNA-FISH results revealed that increased transcript levels of lymphatic vessel-specific markers were significantly associated with a higher density of TLSs in breast cancer. Additionally, lymphatic-specific gene expression was positively correlated with TLS gene scores in two large breast cancer datasets.

conclusionsThese findings highlight the association between TLS density and the axillary residual nodal burden, and illustrate the potential relationship between lymphatic vessels and TLSs in NAC-treated breast cancer patients.