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

Abstract

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.

Figure 1

CD68⁺ (a, b) and CD163⁺ (c, d) macrophages in the sections of LLABCs, using IHC staining, at 200x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to CD68 (Abcam, ab955) at a 1 : 300 dilution for 30 mins at RT, MAbs to CD163 (Abcam, ab74604) at a prediluted concentration for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low level of CD68⁺ and CD163⁺ macrophage infiltration; (b, d) high level of CD68⁺ and CD163⁺macrophage infiltration. Tumours were classified as low level of infiltration when the positively brown membrane-stained cells were scattered or continuous along the tumour margin but did not extend from the tumour front (TF) for more than one cell layer. Extension for two or more layers from the TF was classified as a high level of infiltration.

Figure 2

CD68⁺ (a, b) and CD163⁺ (c, d) macrophages in the sections of axillary lymph nodes (ALNs), using IHC staining, at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to CD68 (Abcam, ab955) at a 1 : 300 dilution for 30 mins at RT, MAbs to CD163 (Abcam, ab74604) at a prediluted concentration for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low percentage of CD68⁺ and CD163⁺ macrophages; (b, d) high percentage of CD68⁺ and CD163⁺macrophages. The positive brown membrane-stained cells in tumour-free medullary areas of ALNs were quantified as the average % of all cells (5 HPFs).

Figure 3

CD1a⁺ DCs (a, b) and CD66b⁺ neutrophils (c, d) in the sections of LLABCs, using IHC staining, at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to CD1a (Dako, M3571) at a 1 : 200 dilution for 15 mins at RT, MAbs to CD66b (LSBio, LS-B7134) at a concentration of 10 μg/ml for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low level of CD1a⁺ DC and CD66b⁺ neutrophil infiltration; (b, d) high level of CD1a⁺ DC and CD66b⁺ neutrophil infiltration. The total number of brown membrane-stained cells, regardless of intensity, in contact with tumour cells or within tumour cell nests (Itu: intratumoural) and in the interstitial stroma (Str: stromal/peritumoural) in 5 HPFs was counted.

Figure 4

CD1a⁺ DCs (a, b) and CD66b⁺ neutrophils (c, d) in the sections of axillary lymph nodes (ALNs), using IHC staining, at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to CD1a (Dako, M3571) at a 1 : 200 dilution for 15 mins at RT, MAbs to CD66b (LSBio, LS-B7134) at a concentration of 10 μg/ml for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low number of CD1a⁺ DCs and CD66b⁺ neutrophils; (b, d) high number of CD1a⁺ DCs and CD66b⁺ neutrophils. The average number of cell counts per HPF in tumour-free paracortical areas of ALNs with the greatest accumulation of the positive brown membrane-stained cells was quantified.

Figure 5

VEGF (a, b) and IDO (c, d) expression in the sections of LLABCs, using IHC staining, at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to VEGF (Dako, M7273) at a 1 : 50 dilution for 30 mins at RT, MAbs to IDO (Abcam, ab55305) at a concentration of 0.75 μg/ml for 15 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low level of expression; (b, d) high level of expression. The H score (% of positive cells (brown membrane/cytoplasmic-stained tumour and immune cells) × intensity of staining (1 to 3)) was used to assess the level of expression; low was ≤100 and high was >100. Scoring performed on a whole tissue section (7–10 HPFs); Tu: tumour, Ma: macrophage, and Ly: lymphocyte.

Figure 6

VEGF (a, b) and IDO (c, d) expression in the sections of axillary lymph nodes (ALNs), using IHC staining at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to VEGF (Dako, M7273) at a 1 : 50 dilution for 30 mins at RT, MAbs to IDO (Abcam, ab55305) at a concentration of 0.75 μg/ml for 15 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low level of expression; (b, d) high level of expression. The H score (% of positive cells (brown membrane/cytoplasmic-stained cells) × intensity of staining (1 to 3)) was used to assess the level of expression; low was ≤100 and high was >100. Scoring performed on nonmetastatic areas of a whole ALN section (7–10 HPFs).