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

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

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

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

The differential contribution of the innate immune system to a good pathological response in the breast and axillary lymph nodes induced by neoadjuvant chemotherapy in women with large and locally advanced breast cancers

The tumour microenvironment consists of malignant cells, stroma, and immune cells. The role of adaptive immunity in inducing a pathological complete response (pCR) in breast cancer with neoadjuvant chemotherapy (NAC) is well studied. The contribution of innate immunity, however, is poorly documented. Breast tumours and axillary lymph nodes (ALNs) from 33 women with large and locally advanced breast cancers (LLABCs) undergoing NAC were immunohistochemically assessed for tumour-infiltrating macrophages (TIMs: M1 and M2), neutrophils (TINs), and dendritic cells (TIDCs) using labelled antibodies and semiquantitative methods. Patients’ blood neutrophils (n = 108), DCs (mDC1 and pDC), and their costimulatory molecules (n = 30) were also studied. Pathological results were classified as pCR, good (GPR) or poor (PRR). In breast and metastatic ALNs, high levels of CD163+ TIMs were significantly associated with a pCR. In blood, high levels of neutrophils were significantly associated with pCR in metastatic ALNs, whilst the % of mDC1 and pDC and expression of HLA-DR, mDC1 CD40, and CD83 were significantly reduced. NAC significantly reduced tumour DCs but increased blood DCs. PPRs to NAC had significantly reduced HLA-DR, CD40, and CD86 expression. Our study demonstrated novel findings documenting the differential but important contributions of innate immunity to pCRs in patients with LLABCs undergoing NAC

Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (TH1, TH2) to immune-mediated tumour cell death induced by NAC

Background The tumour microenvironment consists of malignant cells, stroma and immune cells. In women with large and locally advanced breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC), tumour-infiltrating lymphocytes (TILs), various subsets (effector, regulatory) and cytokines in the primary tumour play a key role in the induction of tumour cell death and a pathological complete response (pCR) with NAC. Their contribution to a pCR in nodal metastases, however, is poorly studied and was investigated. Methods Axillary lymph nodes (ALNs) (24 with and 9 without metastases) from women with LLABCs undergoing NAC were immunohistochemically assessed for TILs, T effector and regulatory cell subsets, NK cells and cytokine expression using labelled antibodies, employing established semi-quantitative methods. IBM SPSS statistical package (21v) was used. Non-parametric (paired and unpaired) statistical analyses were performed. Univariate and multivariate regression analyses were carried out to establish the prediction of a pCR and Spearman’s Correlation Coefficient was used to determine the correlation of immune cell infiltrates in ALN metastatic and primary breast tumours. Results In ALN metastases high levels of TILs, CD4+ and CD8+ T and CD56+ NK cells were significantly associated with pCRs.. Significantly higher levels of Tregs (FOXP3+, CTLA-4+) and CD56+ NK cells were documented in ALN metastases than in the corresponding primary breast tumours. CD8+ T and CD56+ NK cells showed a positive correlation between metastatic and primary tumours. A high % CD8+ and low % FOXP3+ T cells and high CD8+: FOXP3+ ratio in metastatic ALNs (tumour-free para-cortex) were associated with pCRs. Metastatic ALNs expressed high IL-10, low IL-2 and IFN-ϒ. Conclusions Our study has provided new data characterising the possible contribution of T effector and regulatory cells and NK cells and T helper1 and 2 cytokines to tumour cell death associated with NAC in ALNs

Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (TH1, TH2) to immune-mediated tumour cell death induced by NAC

Background and Aim: The tumour microenvironment consists of malignant cells, stroma and immune cells. In women with large and locally advanced breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC), tumour-infiltrating lymphocytes (TILs), various subsets (effector, regulatory) and cytokines in the primary tumour play a key role in the induction of tumour cell death and a pathological complete response (pCR) with NAC. The contribution of the cellular and humoral responses to a pCR in nodal metastases, however, is poorly studied and was investigated. Patients and Methods: Axillary lymph nodes (ALNs) (24 with and 9 without metastases) from women with LLABCs undergoing NAC were immunohistochemically assessed for TILs, T cell subsets, NK cells and cytokine expression using labelled antibodies, employing established semi-quantitative methods. Results: In ALN metastases high levels of TILs, CD4+ and CD8+ T and CD56+ NK cells were significantly associated with pCRs. High levels of TILs and CD56+ NK cells in ALN metastases were independent predictors (univariate analysis) of a pCR with NAC. Significantly higher levels of Tregs (FOXP3+, CTLA-4+) and CD56+ NK cells were documented in ALN metastases than in the corresponding primary breast tumours. CD8+ T and CD56+ NK cells showed a positive correlation between metastatic and primary tumours. A high % CD8+ and low % FOXP3+ T cells and high CD8+ :FOXP3+ ratio in metastatic ALNs (tumour-free paracortex) were associated with pCRs. Metastatic ALNs expressed high IL-10, low IL-2 and IFN-ϒ. Conclusion: Our study has provided new data characterising the possible contribution of T effector/regulatory and NK cells and cytokines to tumour cell death with NAC

Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (TH1, TH2) to immune-mediated tumour cell death induced by NAC

Background and Aim: The tumour microenvironment consists of malignant cells, stroma and immune cells. In women with large and locally advanced breast cancers (LLABCs) undergoing neoadjuvant chemotherapy (NAC), tumour-infiltrating lymphocytes (TILs), various subsets (effector, regulatory) and cytokines in the primary tumour play a key role in the induction of tumour cell death and a pathological complete response (pCR) with NAC. The contribution of the cellular and humoral responses to a pCR in nodal metastases, however, is poorly studied and was investigated. Patients and Methods: Axillary lymph nodes (ALNs) (24 with and 9 without metastases) from women with LLABCs undergoing NAC were immunohistochemically assessed for TILs, T cell subsets, NK cells and cytokine expression using labelled antibodies, employing established semi-quantitative methods. Results: In ALN metastases high levels of TILs, CD4+ and CD8+ T and CD56+ NK cells were significantly associated with pCRs. High levels of TILs and CD56+ NK cells in ALN metastases were independent predictors (univariate analysis) of a pCR with NAC. Significantly higher levels of Tregs (FOXP3+, CTLA-4+) and CD56+ NK cells were documented in ALN metastases than in the corresponding primary breast tumours. CD8+ T and CD56+ NK cells showed a positive correlation between metastatic and primary tumours. A high % CD8+ and low % FOXP3+ T cells and high CD8+ :FOXP3+ ratio in metastatic ALNs (tumour-free paracortex) were associated with pCRs. Metastatic ALNs expressed high IL-10, low IL-2 and IFN-ϒ. Conclusion: Our study has provided new data characterising the possible contribution of T effector/regulatory and NK cells and cytokines to tumour cell death with NAC

Additional file 1: Table S3. of Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (Th1, Th2) to immune-mediated tumour cell death induced by NAC

Patient and Tumour Characteristics, Responses to Neoadjuvant Chemotherapy (n = 33). (DOCX 56 kb