Evaluation of immune cell infiltrates and expression of cytokines/biological molecules in the microenvironment of tumours and tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy: crucial contribution to immune-mediated tumour cell death

Abstract

Background Neoadjuvant chemotherapy (NAC) is being used as first line treatment in women with large and locally advanced breast cancers (LLABCs). However, the response to NAC is difficult to predict. Growing evidence suggests that these patients are immunosuppressed and that circulating immunosuppressive regulatory cells and humoral factors affect the response to NAC. We explored the possible role of the in situ tumour immune milieu in inducing and affecting the responses to NAC, and the contribution of concomitant systemic circulating regulatory cells. Methods Paraffin-embedded breast cancers and ipsilateral axillary lymph nodes (ALNs) from pre- and post-NAC samples of a cohort of 33 women with LLABCs, 16 of whom had their blood regulatory cells previously investigated. Various immune cell infiltrations and expression of cytokines/ biological molecules in the specimens were studied using appropriate monoclonal antibodies and immunohistochemistry. Statistical analysis was carried out using non-parametric tests with SPSS version 21. Results High levels of pre-NAC tumour-infiltrating lymphocytes (TILs) (p<0.001) and subsets of CD4⁺T cells (intratumoural, p=0.023; peritumoural, p=0.001), CD8+T cells (intratumoural, p=0.008; peritumoural, p=0.002) and CD56⁺NK cells (intratumoural, p=0.001; peritumoural, p<0.001) were significantly associated with a pathological complete response (pCR). High levels of CD163⁺macrophages were also significantly associated with a good pathological response (p=0.004) and pCR (p=0.008). There was a positive correlation between the CD8:FOXP3 ratio and grade of pathological response. In multivariate analyses, TILs and peritumoural CD56+NK cells were found to be independent predictive factors for pCR. There was a significantly high expression of IL-10 in post-NAC breast specimens with poor responses to NAC (p<0.001). NAC significantly reduced infiltrating T regulatory cells (Tregs) (p=0.001) and PD1⁺T cells (p=0.005), as well as expression of IL-4 (p=0.016). There was no significant difference between the percentages (%) of immune cells present in ALNs with or without metastases but there was a T helper-2 cytokine polarisation in metastatic ALNs. Metastatic ALNs with a high % of CD8+T cells (p=0.048) and low % of FOXP3+Tregs (p=0.019) were significantly associated with an ALN pCR. There was a significantly positive correlation between circulating and intratumoural infiltrating Tregs following NAC (p=0.003). Conclusions The tumour immune microenvironment is a key factor in achieving a good pathological response with NAC. Tumour and blood immune parameters may be clinically useful in identifying women with LLABCs likely to respond to NAC. Our findings also suggest that the beneficial effects of NAC are mediated via modulation of anticancer immunity, in particular by reduction of T regulatory cells and immunosuppressive humoral factors