Quantitative multiplex immunofluorescence analysis identifies infiltrating PD1+CD8+ and CD8+ T cells as predictive of response to neoadjuvant chemotherapy in breast cancer

Background This study aimed to explore the potentially predictive role and dynamic changes of immune checkpoints on T cell subsets in patients with breast cancer receiving neoadjuvant chemotherapies. Methods Fluorescent multiplex immunohistochemistry (mIHC) was used to stain CD4, CD8, PD1, TIM3, and cytokeratins simultaneously in paired breast cancer samples before and after neoadjuvant therapies (NAT) in a prospective cohort (n = 50). Singleplex IHC was conducted to stain for CD3 in 100 cases with inclusion of extra retrospective 50 cases. Cell levels were correlated with clinicopathological parameters and pathological complete response (pCR). Results In pretreatment tumors, the percentages of infiltrating CD8+, PD1+, PD1+CD8+, and the ratio of PD1+CD8+/CD8+ cells, were higher in pCR than non‐pCR patients in either the stromal or intratumoral area, but PD1+CD4+, TIM3+CD4+, TIM3+CD8+ cells and CD4+/CD8+ ratio was not. Multivariate analyses showed that the percentage of intratumoral CD8+ cells (OR, 1.712; 95% CI: 1.052–2.786; P = 0.030) and stromal PD1+CD8+/CD8+ ratio (OR, 1.109; 95% CI: 1.009–1.218; P = 0.032) were significantly associated with pCR. Dynamically, reduction in the percentages of PD1+, CD8+ and PD1+CD8+ cells after therapy strongly correlated with pCR. Notably, incremental percentages of PD1+CD8+ cells, rather than TIM3+CD8+, were shown in tumors from non‐pCR patients after NAT. CD3 staining confirmed the percentage of T cells were associated with pCR. Conclusions PD1+CD8+ rather than TIM3+CD8+ cells are main predictive components within tumor‐infiltrating T cells in NAT breast cancer patients. Dynamically incremental levels of PD1+CD8+ cells occurred in non‐pCR cases after NAT, suggesting the combination of chemotherapy with PD1 inhibition might benefit these patients. Key points Significant findings of the study PD1+CD8+, rather than TIM3+CD8+, T cells are the main component to predict the response of neoadjuvant therapies in breast cancer. What this study adds Incremental levels of PD1+CD8+ T cells in non‐pCR post‐NAT tumors suggest PD1 inhibition might benefit in the neoadjuvant setting.