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 (
Anticancer immune mechanisms play an important role in the induction, development, and dissemination of malignant disease in man [
The role of adaptive immunity (T effector and regulatory cells, T helper (Th), and suppressor cytokines) in women with breast cancer undergoing NAC has been investigated in primary breast tumours, but much less so in tumour-draining ALNs. Significant associations have been documented between high levels of infiltration by T effector cells (CD4+ and CD8+) and low levels of T regulatory cells (Tregs: FOXP3+ (forkhead box protein 3) and CTLA-4+ (cytotoxic T lymphocyte antigen-4)) and subsequent pCRs with NAC in primary breast tumours and ALN metastases [
We have recently described the key role played by NK cells within primary breast tumours and tumour-draining ALNs. Our studies have shown a significant association between high levels of tumour-infiltrating CD56+ NK cells, in both the primary tumours and ALN metastases, and subsequent pCRs with NAC. These findings highlight the important contribution of innate immunity to immune-mediated tumour cell death in patients with breast cancer undergoing NAC.
Macrophages are terminally differentiated myeloid cells, closely related to DCs, and are resident in tissues. Human solid tumours recruit macrophages into their microenvironment. Breast cancers, in particular, contain a substantial number of macrophages [
Polymorphonuclear leukocytes (neutrophils) play a crucial role in dealing with invading pathogens and assist in wound healing. They release a range of toxic substances (reactive oxygen species and proteinases) to deal with invading microorganisms [
Dendritic cells (DCs) are derived from haematopoietic progenitor cells in the bone marrow, monocytes being major precursor cells. They are crucial antigen-presenting cells, initiating and directing adaptive immune responses [
Various factors, including tumour stroma-released chemokines and cytokines (CCL19 and transforming growth factor-
We have recently documented, in women with LLABCs undergoing NAC, that a significant reduction of both circulating and tumour-infiltrating Tregs (FOXP3+, CTLA-4+) and high levels of TILs and CD8+ T cells in the primary and ALN metastatic tumours were significantly associated with subsequent pCRs [
Paraffin-embedded sections of breast tumours and tumour-draining ALNs from 33 women with large (L: ≥3 cm) and locally advanced breast cancers (LABCs: T3, 4; N1, 2; M0), enrolled in a study of NAC (108 patients enrolled between 2008 and 2011), were studied [
The NAC trial evaluated the effect of the addition of capecitabine (X) to docetaxel (T) preceded by adriamycin and cyclophosphamide (AC). Pathological responses were assessed in the excised surgical specimens after NAC. Established and previously published grading criteria were used to define histopathological responses in the breast [
The study was given approval by the Leicestershire, Northamptonshire & Rutland Research Ethics Committee 1: Reference number 07/H0406/260; Favourable Opinion 24/01/2008. All patients enrolled in the study gave informed consent to participate in and to publish the results of the study. The study registration is
Venous blood samples were collected before (
The mDC1high (Lin1−, HLA-DR+, CD11c+, and CD1c+) and the pDChigh (Lin1−, HLA-DR+, CD11c+, and CD303+) subsets and their costimulatory molecules were documented in 2 × 106 cells/100
Blood neutrophil counts (pre- and post-NAC) in 108 women with LLABC were collected and used in the analysis to evaluate association with NAC-induced pCR in the breast and tumour-draining ALNs.
Immunohistochemical assessments of immune cell subsets and expression of indoleamine 2,3-dioxygenase (IDO) and vascular endothelial growth factor (VEGF) were performed in 4
Whole tissue sections were studied rather than microarrays (to minimise sampling bias). The majority of macrophages present in breast tumours were located along the border of tumour nests. Immunostaining for TIMs was evaluated along the tumour front (TF) over the whole section (7–10 view fields per section). This evaluation followed previously published studies documenting the level of TIMs [
To evaluate the presence and extent of CD1a+ and CD66b+ cells in the breast tumours, the average numbers of brown membrane-stained cells regardless of the intensity were counted in 5 high-power fields (400x HPFs). Positively stained cells in contact with tumour cells or within the tumour cell nests were defined as “intratumoural” whereas positively stained cells in the interstitial stroma surrounding tumour nests were defined as “peritumoural/stromal.” Evaluation of infiltrations in post-NAC specimens was undertaken on residual tumour nests, and in the case of pCR (complete disappearance of invasive tumour cells in the specimen), in the tumour bed. The latter was characterised histologically as a hyalinised, amorphous area with haemosiderin deposits [
Positively stained macrophages (CD68+ and CD163+) in ALNs were quantified as the average % of all cells in 5 HPFs in nonmetastatic medullary areas of ALNs. The average number of cell counts in 5 HPFs in nonmetastatic areas with the greatest accumulations of positively stained cells on scanning at low magnification was determined for CD66b+ and CD1a+ cells. These quantitative evaluations followed the methodological scoring for documenting various immune cell subsets present in ALNs in patients with breast cancer [
To evaluate the presence of IDO and VEGF, the semiquantitative H scoring system was employed using whole tissue sections. The H score was calculated by multiplying the % of positive cells (tumour and immune) by a factor representing the intensity of immune reactivity (1 for weak, 2 for moderate, and 3 for strong), giving a maximum score of 300. A score of <50 was considered negative, and a score of 50–100 was considered weakly positive (1+). A score of 101–200 was regarded as moderately positive (2+) and a score of 201–300 as strongly positive (3+). Negative and 1+ were considered as low expression whereas 2+ and 3+ were considered as high expression [
All scored assessments were performed without the knowledge of the patients’ clinical and pathological parameters. The pathological responses were assessed by a consultant breast pathologist. The scoring systems used in establishing the biological markers present in breast tumours and metastatic tumours in ALNs, and ALNs in this investigation, followed previously published studies documenting the scoring systems employed (described above).
Statistical analyses were performed with the IBM SPSS statistics software, version 21 (SPSS Inc., Chicago, IL, USA). Where the data did not follow a normal distribution, a nonparametric test (Mann–Whitney
The sample size of this study was based on a cohort of patients from a previous study in which circulating Tregs were documented pre- and post-NAC [
The patient and tumour characteristics of the 33 patients studied are shown in Table A (Supplementary Table available online at
High levels of CD163+ TIMs in breast cancers were significantly associated with a GPR and pCR (
Analyses of tumour-infiltrating CD68+ and CD163+ macrophages in the breast tumours in women with LLABCs and subsequent PCR following NAC.
Macrophages | Groups | Pre-NAC | |||
---|---|---|---|---|---|
Low infiltration ( |
High infiltration ( |
Pearson chi-square value (GPR versus PPR, PCR versus non-PCR) |
| ||
CD68+ ( |
Good pathological response (GPR, |
5 | 4 | 1.667 | 0.197 |
Poor pathological response (PPR, |
6 | 1 | |||
Pathological complete response (PCR, |
3 | 3 | 1.571 | 0.210 | |
Nonpathological complete response (non-PCR, |
8 | 2 | |||
|
|||||
CD163+ ( |
Good pathological response (GPR, |
5 | 16 | 8.192 | 0.004 |
Poor pathological response (PPR, |
9 | 3 | |||
Pathological complete response (PCR, |
3 | 13 | 7.127 | 0.008 |
|
Nonpathological complete response (non-PCR, |
11 | 6 |
LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy;
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.
Eight cycles of NAC, on the other hand, did not alter the level of infiltration by CD68+ and CD163+ TIMs in post-NAC tumour specimens when compared with pre-NAC specimens (Table
Alteration of tumour-infiltrating CD68+ and CD163+ macrophages in the breast tumours in women with LLABCs undergoing NAC.
Macrophages | Groups | Post-NAC |
| ||
---|---|---|---|---|---|
Low infiltration ( |
High infiltration ( | ||||
CD68+ ( |
Pre-NAC | Low infiltration ( |
10 | 1 | 0.375 |
High infiltration ( |
4 | 1 | |||
|
|||||
CD163+ ( |
Pre-NAC | Low infiltration ( |
4 | 2 | 0.289 |
High infiltration ( |
6 | 4 |
LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (3)related sample McNemar test.
High levels of CD163+ TIMs in metastatic deposits in tumour-draining ALNs were significantly associated with a pCR (
Analyses of CD163+ macrophages in metastatic tumours in ALNs in women with LLABCs and subsequent PCR following NAC.
Groups | Pre-NAC | ||||
---|---|---|---|---|---|
Low infiltration ( |
High infiltration ( |
Pearson chi-square value (PCR versus non-PCR) |
| ||
CD163+ macrophages ( |
Pathological complete response (PCR, |
0 | 9 | 8.811 | 0.003 |
Nonpathological complete response (non-PCR, |
7 | 4 |
ALNs: axillary lymph nodes; LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy;
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).
Comparison of primary breast cancers with ALN metastases showed no significant differences in the levels of infiltration by CD163+ TIMs (Table
Comparison of CD163+ macrophages between primary breast tumours and ALN metastatic tumours in women with LLABCs.
Groups | Metastatic tumours in ALNs |
| |||
---|---|---|---|---|---|
Low infiltration ( |
High infiltration ( | ||||
CD163+ macrophages | Primary tumours in breast | Low infiltration ( |
6 | 2 | 1.000 |
High infiltration ( |
1 | 11 |
ALN: axillary lymph node; LLABCs: large and locally advanced breast cancers; (3)related sample McNemar test.
Neither good pathological responders nor those whose tumours had a pCR with NAC had a significant association with pre-NAC CD1a+ TIDCs or CD66b+ TINs. The infiltration was assessed both intratumourally and peritumourally (stroma) (Table
Analyses of tumour-infiltrating CD1a+ dendritic cells and CD66b+ neutrophils in the breast tumours in women with LLABCs and subsequent PCR following NAC.
Cell subsets | Groups | Pre-NAC intratumoural median (range)(3) |
|
Pre-NAC peritumoural median (range)(3) |
|
---|---|---|---|---|---|
CD1a+ ( |
Good pathological response (GPR, |
3 (1–104) | 0.837 | 1 (1–16) | 0.837 |
Poor pathological response (PPR, |
11 (0–63) | 2 (0–11) | |||
Pathological complete response (PCR, |
3 (1–104) | 0.713 | 1.5 (1–16) | 0.492 | |
Nonpathological complete response (non-PCR, |
4 (0–63) | 1.5 (0–11) | |||
|
|||||
CD66b+ ( |
Good pathological response (GPR, |
2 (0–53) | 0.174 | 2 (0–71) | 0.408 |
Poor pathological response (PPR, |
1 (0–3) | 1 (0–2) | |||
Pathological complete response (PCR, |
3 (0–53) | 0.181 | 5 (0–71) | 0.118 | |
Nonpathological complete response (non-PCR, |
1 (0–3) | 1 (0–2) |
LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (3)total cell count per 5 high-power fields (core biopsies of breast cancers); (4)Mann–Whitney
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
Alteration of tumour-infiltrating CD1a+ dendritic cells and CD66b+ neutrophils in breast tumours in women with LLABCs undergoing NAC.
Groups | Pre-NAC median (range)(3) | Post-NAC median (range)(3) |
| |
---|---|---|---|---|
CD1a+ | Intratumoural infiltration ( |
3.5 (0–104) | 0 (0–2) | 0.001 |
Peritumoural infiltration ( |
1.5 (0–16) | 1 (0–7) | 0.184 | |
|
||||
CD66b+ | Intratumoural infiltration ( |
1 (0–53) | 4.5 (0–50) | 0.125 |
Peritumoural infiltration ( |
1.5 (0–71) | 2.5 (0–82) | 0.470 |
LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (3)cell count in 400x HPF; (4)Wilcoxon signed-rank test;
There was no significant difference in the level of macrophages (CD68+ and CD163+) in tumour-draining ALNs, comparing metastatic (tumour-free areas) with nonmetastatic, in women with LLABCs undergoing NAC (Table
Analyses of immune cell subsets in ALNs in women with LLABCs undergoing NAC.
Immune cell subsets | Groups | ALN median (range)(4) |
|
---|---|---|---|
CD68+ macrophages ( |
Nonmetastatic ALNs ( |
25.0 (14.8–34.0) | 0.918 |
Metastatic ALNs ( |
29.0 (13.8–33.0) | ||
CD163+ macrophages ( |
Nonmetastatic ALNs ( |
21.0 (16.0–29.0) | 1.000 |
Metastatic ALNs ( |
23.0 (10.0–33.0) | ||
CD1a+ DCs ( |
Nonmetastatic ALNs ( |
12.8 (0.8–62.0) | 0.536 |
Metastatic ALNs ( |
23.8 (6.6–67.0) | ||
CD66b+ PMNs ( |
Nonmetastatic ALNs ( |
5.2 (0.6–94.0) | 0.837 |
Metastatic ALNs ( |
8.4 (1.0–163.0) |
ALNs: axillary lymph nodes; LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (4)average percentage of positively stained cells out of all the lymphoid cells in the ALN sections examined for CD68+ and CD163+ macrophages, average cell count of positively stained cells per 400x high-power field in the ALN sections examined for CD1a+ DCs and CD66b+ PMNs; (5)Mann–Whitney
The presence of CD66b+ neutrophils and CD1a+ DCs in ALNs in tumour-free paracortical areas of ALNs is illustrated in Figures
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
High levels of expression of VEGF in primary breast cancers were significantly associated with a pCR (
Analyses of VEGF and IDO expression in the breast tumours in women with LLABCs (pre-NAC and post-NAC) and association with a PCR.
VEGF/IDO ( |
Groups | Pre-NAC | Post-NAC | ||||||
---|---|---|---|---|---|---|---|---|---|
Low/negative expression ( |
High expression ( |
Pearson chi-square value (GPR versus PPR, PCR versus non-PCR) |
|
Low/negative expression ( |
High expression ( |
Pearson chi-square value (GPR versus PPR, PCR versus non-PCR) |
| ||
VEGF | Good pathological response (GPR, |
5 | 4 | 1.667 | 0.197 | 7 | 2 | 0.780 | 0.377 |
Poor pathological response (PPR, |
6 | 1 | 4 | 3 | |||||
Pathological complete response (PCR, |
2 | 4 | 5.605 | 0.018 |
5 | 1 | 0.950 | 0.330 | |
Nonpathological complete response (non-PCR, |
9 | 1 | 6 | 4 | |||||
|
|||||||||
IDO | Good pathological response (GPR, |
6 | 3 | 0.042 | 0.838 | 6 | 3 | 0.152 | 0.696 |
Poor pathological response (PPR, |
5 | 2 | 4 | 3 | |||||
Pathological complete response (PCR, |
3 | 3 | 1.571 | 0.210 | 4 | 2 | 0.071 | 0.790 | |
Nonpathological complete response (non-PCR, |
8 | 2 | 6 | 4 |
VEGF: vascular endothelial growth factor; IDO: indoleamine 2,3-dioxygenase; LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy;
No alteration of expression of IDO and VEGF in the breast tumours in women with LLABCs undergoing NAC.
VEGF/IDO ( |
Groups | Post-NAC |
|
||
---|---|---|---|---|---|
Low/negative expression ( |
High expression ( | ||||
VEGF | Pre-NAC | Low/negative expression ( |
8 | 3 | 1.000 |
High expression ( |
3 | 2 | |||
|
|||||
IDO | Pre-NAC | Low/negative expression ( |
8 | 3 | 1.000 |
High expression ( |
2 | 3 |
IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (5)related sample McNemar Test.
Figure
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
There was no difference in the expression of IDO and VEGF in ALNs, whether metastatic (tumour-free paracortical area) or nonmetastatic (data not shown).
Figure
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
High levels of CD163+ TIMs were significantly associated with ER status (
Clinical and pathological parameters of patients (
Groups | TIMs | PCR | ||||||
---|---|---|---|---|---|---|---|---|
Low infiltration ( |
High infiltration ( |
Pearson chi-square value |
|
Non-PCR ( |
PCR ( |
Pearson chi-square value |
| |
Age (years) | ||||||||
<50 | 6 | 8 | 0.002 | 0.966 | 8 | 6 | 0.308 | 0.579 |
≥50 | 8 | 11 | 9 | 10 | ||||
BMI (kg/m2) | ||||||||
≤30 | 10 | 10 | 1.193 | 0.275 | 11 | 9 | 0.247 | 0.619 |
>30 | 4 | 9 | 6 | 7 | ||||
Menopausal status | ||||||||
Pre | 5 | 11 | 1.588 | 0.208 | 8 | 8 | 0.029 | 0.866 |
Post | 9 | 8 | 9 | 8 | ||||
Tumour size | ||||||||
<40 mm | 8 | 10 | 0.066 | 0.797 | 9 | 9 | 0.036 | 0.849 |
≥40 mm | 6 | 9 | 8 | 7 | ||||
Nodal status | ||||||||
Negative | 5 | 5 | 0.337 | 0.561 | 5 | 5 | 0.013 | 0.909 |
Positive | 9 | 14 | 12 | 11 | ||||
Tumour grade | ||||||||
1 (low) | 1 | 1 | 11.270 | 0.004 |
2 | 0 | 9.303 | 0.010 |
2 (moderate) | 10 | 3 | 10 | 3 | ||||
3 (high) | 3 | 15 | 5 | 13 | ||||
Oestrogen receptor | ||||||||
Negative | 2 | 9 | 3.970 | 0.046 |
3 | 8 | 3.882 | 0.049 |
Positive | 12 | 10 | 14 | 8 | ||||
HER-2 receptor | ||||||||
Negative | 10 | 13 | 0.035 | 0.853 | 13 | 10 | 0.762 | 0.383 |
Positive | 4 | 6 | 4 | 6 | ||||
NAC regimen | ||||||||
AC-TX | 6 | 10 | 0.308 | 0.579 | 6 | 10 | 2.443 | 0.118 |
AC-T | 8 | 9 | 11 | 6 | ||||
Recurrent disease(4) | ||||||||
No | 8 | 14 | 0.992 | 0.319 | 7 | 15 | 10.252 | 0.001 |
Yes | 6 | 5 | 10 | 1 | ||||
Death(4) | ||||||||
No | 11 | 16 | 0.172 | 0.678 | 12 | 15 | 2.972 | 0.085 |
Yes | 3 | 3 | 5 | 1 |
NAC: neoadjuvant chemotherapy; BMI: body mass index (≤30: nonobese, >30: obese); AC-TX: doxorubicin, cyclophosphamide, taxotere, and xeloda® (capecitabine), respectively; (4)4-year follow-up;
High levels of circulating PMN neutrophils pre-NAC in women with LLABCs (
Analyses of blood PMN neutrophils in women with LLABC and specific clinical and pathological parameters.
Groups | Pre-NAC median (range)(3) |
|
Post-NAC median (range)(3) |
| |
---|---|---|---|---|---|
Blood PMNs | Good pathological response (GPR, |
4.13 (2.15–11.30) | 0.796 | 3.05 (1.40–6.75) | 0.134 |
Poor pathological response (PPR, |
4.07 (1.80–10.10) | 3.47 (0.03–8.73) | |||
Pathological complete response (PCR, |
4.50 (2.15–11.30) | 0.381 | 3.10 (1.40–6.75) | 0.755 | |
Nonpathological complete response (non-PCR, |
3.94 (1.80–10.10) | 3.26 (0.03–8.73) | |||
Nodal metastasis ( |
3.94 (1.80–10.10) | 0.634 | 3.11 (0.03–8.26) | 0.337 | |
No nodal metastasis ( |
4.16 (2.15–11.30) | 3.36 (1.54–8.73) | |||
Nodal pCR ( |
5.43 (2.84–10.10) | 0.002 |
3.20 (1.40–8.26) | 0.892 | |
No nodal pCR ( |
3.65 (1.80–9.17) | 3.05 (0.03–6.37) | |||
Recurrent disease(5) ( |
4.02 (1.80–8.70) | 0.612 | 3.19 (0.03–5.80) | 0.878 | |
No recurrent disease ( |
4.10 (2.15–11.30) | 3.23 (1.33–8.73) | |||
Death(5) ( |
3.87 (2.23–6.64) | 0.276 | 3.00 (0.03–5.42) | 0.360 | |
Survive ( |
4.11 (1.80–11.30) | 3.23 (1.33–8.73) |
LLABC: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (3)×109 cells/litre; (4)Mann–Whitney
Alteration of blood PMN neutrophils in women with LLABCs undergoing NAC.
Group | Pre-NAC median (range)(3) | Post-NAC median (range)(3) |
|
---|---|---|---|
Blood PMNs ( |
4.09 (1.80–11.30) | 3.20 (0.03–8.73) | <0.001 |
LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; (3)×109 cells/litre; (4)Wilcoxon signed-rank test;
The % of DCs in the blood of women with LLABCs (
Percentage of DC subsets and expression of costimulatory and LNH molecules in the circulation of women with LLABCs.
mDC1s and pDCs | % of subsets and costimulatory molecules in women with LLABCs ( |
% of subsets and costimulatory molecules in HFDs ( |
|
---|---|---|---|
Lin1−, HLA-DR+ DCs | 1.26 ± 0.20 | 1.70 ± 0.30 | 0.034 |
mDC1high (Lin1−, HLA- DR+, CD11c+CD1c+) | 0.10 ± 0.04 | 0.15 ± 0.05 | 0.045 |
pDChigh (Lin1−, HLA-DR+, CD11c+CD303+) | 0.03 ± 0.02 | 0.06 ± 0.02 | 0.041 |
mDC1 HLA-DR |
47.30 ± 8.00 | 65.00 ± 3.10 | 0.045 |
pDC HLA-DR |
48.12 ± 9.50 | 65.52 ± 5.00 | 0.002 |
mDC1 CD40 |
35.01 ± 8.00 | 44.87 ± 6.50 | 0.011 |
mDC1 CD80 |
1.40 ± 0.60 | 1.87 ± 0.35 | NS |
mDC1 CD83 |
3.18 ± 0.40 | 4.04 ± 0.30 | 0.047 |
mDC1 CD86 |
28.56 ± 3.00 | 34.07 ± 8.00 | NS |
mDC1 CD197 |
20.35 ± 4.50 | 24.68 ± 9.00 | NS |
LNH: lymph node homing; LLABCs: large and locally advanced breast cancers; mDC1: myeloid dendritic cell; pDC: plasmacytoid dendritic cell; HFDs: healthy female donors; NS: nonsignificant;
The expression of the costimulatory molecule HLA-DR was significantly reduced in both the blood mDC1+ and pDC+ subsets, compared with HFDs (47.30 ± 8.00% versus 65.00 ± 3.10% (
The expression of CD40 and CD83 was also depressed in mDC1shigh, compared with HFDs (35.01 ± 8.00% versus 44.87 ± 6.50% (
Before NAC, the % baseline levels of DCs in women who subsequently had a good pathological response with NAC (
Effect of NAC on blood DC levels in women with LLABCs.
Pathological responders | DC baseline levels (%) |
| |
|
|
GPRs + PPRs | 1.34 ± 0.30 |
GPRs + PPRs versus HFDs | NS |
GPRs | 1.64 ± 0.25 |
GPRs versus HFD | NS |
PPRs | 1.29 ± 0.25 |
PPRs versus HFDs | NS |
|
|
Pathological responders | DC post-NAC levels (%) |
| |
|
|
GPRs | 3.24 ± 1.62 |
GPRs versus HFDs | 0.024 |
PPRs | 2.17 ± 0.5 |
PPRs versus HFDs | NS |
NAC: neoadjuvant chemotherapy; LLABCs: large and locally advanced breast cancers; GPRs: good pathological responders (pCR or ≥90% reduction of invasive disease); PPRs: poor pathological responders (no response or <90% reduction of invasive disease); HFDs: healthy female donors; NS: nonsignificant;
After 8 cycles of NAC, those women whose breast cancers showed a PPR had significantly lower levels of blood mDC1shigh (
Percentage of DC subsets and expression of HLA-DR in the blood of women with LLABC undergoing NAC. Baseline (B) levels in LLABCs versus completion of chemotherapy (CC) levels in different responders.
Study group comparisons | mDC1 | mDC1 | pDC | pDC | |
---|---|---|---|---|---|
HLA-DR | HLA-DR | ||||
Good pathological responders (GPRs: |
B (%) | 0.12 ± 0.04 | 36.07 ± 6.00 | 0.02 ± 0.02 | 50.86 ± 10.5 |
CC (%) | 0.10 ± 0.04 | 46.33 ± 8.00 | 0.05 ± 0.02 | 58.05 ± 12.00 | |
B versus CC |
NS | NS | NS | NS | |
CC versus HFDs |
NS | NS | NS | NS | |
|
|||||
Poor pathological responders (PPRs: |
B (%) | 0.08 ± 0.03 | 31.29 ± 6.00 | 0.02 ± 0.02 | 53.21 ± 10.00 |
CC (%) | 0.05 ± 0.03 | 29.83 ± 5.00 | 0.03 ± 0.02 | 49.97 ± 6.00 | |
B versus CC |
NS | NS | NS | NS | |
CC versus HFDs |
0.048 |
0.001 |
0.017 |
0.023 |
|
|
|||||
Post-NAC GPR versus PPR | GPR CC versus PPR CC |
NS | 0.041 |
NS | NS |
LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; GPRs: complete or ≥90% reduction of tumour cell mass; PPRs: no or ≤90% reduction of tumour cell mass; HFDs: healthy female donors; NS: nonsignificant;
After 8 cycles of NAC, women whose breast cancers showed a PPR also had significantly lower levels of expression of mDC1 CD40 (
Expression (%) of costimulatory and LNH molecules on mDC1s in the blood of women with LLABCs undergoing NAC. Baseline (B) levels in LLABCs versus completion of chemotherapy (CC) levels in different responders.
Study group comparisons | CD40 | CD80 | CD83 | CD86 | CD197 | |
---|---|---|---|---|---|---|
Good pathological responders (GPR: |
B (%) | 40.60 ± 10.00 | 2.02 ± 1.30 | 4.45 ± 0.75 | 40.97 ± 14.00 | 31.29 ± 8.00 |
CC (%) | 43.13 ± 10.00 | 3.83 ± 2.00 | 3.71 ± 1.70 | 28.66 ± 7.00 | 48.34 ± 14.00 | |
B versus CC |
NS | NS | NS | NS | NS | |
CC versus HFDs |
NS | NS | NS | NS | NS | |
|
||||||
Poor pathological responders (PPRs: |
B (%) | 26.80 ± 10.00 | 1.48 ± 0.50 | 2.98 ± 2.00 | 21.37 ± 10.00 | 22.73 ± 6.00 |
CC (%) | 16.80 ± 8.00 | 2.29 ± 1.20 | 4.47 ± 1.80 | 6.81 ± 6.00 | 13.83 ± 5.00 | |
B versus CC |
NS | NS | NS | NS | NS | |
CC versus HFDs |
0.001 |
NS | NS | 0.001 |
NS | |
|
||||||
Post-NAC GPR versus PPR | GPR CC versus PPR CC |
0.005 |
NS | NS | 0.004 |
0.003 |
LNH: lymph node homing; LLABCs: large and locally advanced breast cancers; NAC: neoadjuvant chemotherapy; GPRs: complete or >90% reduction of tumour cell mass; PPRs: no or ≤90% reduction of tumour cell mass; HFDs: healthy female donors; NS: nonsignificant;
Comparison of blood levels of mDC1 expression of costimulatory and LNH molecules between GPRs and PPRs showed a significant reduction in CD40, CD86, and CD197 expression on circulating mDC1s (
These findings demonstrate the significant association between the reduced % (mDC1s and pDCs) and expression of HLA-DR (mDC1s and pDCs), CD40, and CD86 (mDC1s) and failure to achieve a GPR with NAC. Thus, women whose breast cancers failed to undergo a GPR with NAC showed significantly reduced, inactive, and immature DC subsets in the circulation.
In the current study reported, the CD163+ TIMs (M2 macrophage phenotype) in both the primary breast cancer and metastatic ALNs were significantly associated with pCRs in malignant tissue following NAC. TIMs are derived from circulating monocytes and primitive bone marrow progenitors [
A pCR following NAC in breast cancer has been shown in some studies to be a surrogate marker for a good prognosis and survival [
M1 TIMs are activated by interferon-
In sentinel lymph nodes (SLNs), Mansfield et al. found that the presence of sinusoidal CD163+ M2 macrophages was associated with a favourable nodal status in patients with breast cancer. They did not investigate TIMs in metastatic SLNs [
In patients with cancer, blood neutrophils tend to be immature and produce low levels of free radicals [
There were significantly reduced blood levels of PMN neutrophils following NAC, albeit all patients were given granulocyte colony-stimulating factor following randomisation.
Tumour entry of PMN neutrophils is induced by chemotactic molecules secreted by intratumoural TIMs and cancer cells [
In tumour-draining metastatic ALNs, there was also no significant association between the level of infiltration by CD66b+ TINs and pCR with NAC. Moreover, there was no difference in the level of infiltration by CD66b+ neutrophils in the paracortical areas of metastatic (tumour-free areas) and nonmetastatic ALNs. Such findings have not previously been documented.
In our study, we investigated tumour-infiltrating CD1a+ DCs and concurrently circulating DCs, mDC1shigh and pDCshigh. In solid cancers, evidence suggests that DCs in the tumour-microenvironment are present in small numbers, are immature, and poorly activated [
Although the presence of TIDCs has been documented to be associated with a better clinical outcome in a number of human solid cancers, this is not the case with CD1a+ TIDCs in breast cancer [
The percentages of circulating levels of DCs, mDC1s and pDCs, were significantly reduced in women with LLABCs when compared with healthy females. In addition, the expression of HLA-DR, CD40, and CD83 molecules on the surface of mDC1s were also significantly reduced, as was HLA-DR expression of pDCs. The expression of the costimulatory molecules CD80/86 was unchanged compared with healthy females. We did not carry out any functional assays to ascertain specific effector functions such as efficacy in antigen presentation and secretion of cytokines. NAC, in particular anthracyclines and taxanes, is known to disrupt tumour cells, exposing expression of calreticulin and release of tumour-associated antigens (TAAs) [
There was a significant difference in the level of circulating DCs between those patients whose tumours showed a good pathological response (GPR; pCR or ≥90% loss of tumour cell mass) and those whose tumours had a poor pathological response (PPR; no or <90% loss of tumour cell mass) to 8 cycles of NAC. Following NAC, the blood DC levels in the GPR group were increased and were significantly higher than the levels documented in healthy females. There was also a significantly reduced % of circulating mDC1shigh and pDCshigh and reduced expression of HLA-DR, CD40, and CD83 on mDC1shigh and HLA-DR on pDCshigh in those patients whose tumours showed a PPR to NAC. In addition, post-NAC patients whose tumours had a GPR had a significantly increased expression of HLA-DR, CD40, CD86, and the LNHR CD197. These significant associations suggest a possible and important interaction between DCs and TAA presentation to naïve T cells and immune-induced tumour cell death during NAC. The findings documented, to the best of our knowledge, have not been published previously.
Many chemotherapeutic drugs produce short-lived inhibitory effects on innate and adaptive immune cells. Some (anthracyclines, taxanes, cyclophosphamide, capecitabine, and gemcitabine), however, can enhance or suppress specific aspects of the immune mechanism and activate immune-mediated tumour cell death, contributing to the good pathological responses documented in primary cancers and metastatic ALNs [
Our study has provided further knowledge and understanding of the relevance and contribution of certain components of innate immunity to tumour cell death in both the primary breast cancer and metastases in tumour-draining ALNs in women with LLABCs undergoing NAC. High levels of TIMs (M2) appear to induce/enhance pCRs in primary and ALN metastatic breast cancers probably through their association with high tumour grade and negative ER status. High level of expression of VEGF and the resultant increased vascularity results in enhanced delivery of chemotherapeutic agents to the tumour cell milieu. Circulating levels of DCs and expression of HLA-DR and costimulatory molecules were significantly reduced in patients with LLABCs. This diminished number of activated DCs and thus decreased capacity to present TAAs (released by NAC and innate NK cells) to naïve adaptive T cells results in reduced generation of CTLs. This trend was significantly reversed in patients in whom NAC induced a pCR. These various immune mechanisms highlight the close and important interaction between innate and adaptive anticancer immunity.
There is a significant body of evidence documenting the important contribution by circulating and tumour-infiltrating T effector (CD4+ and CD8+) and regulatory (FOXP3+ and CTLA-4+) cells and NK cells in immune-mediated breast cancer cell death in women with LLABCs undergoing NAC. Our novel findings have further increased our knowledge and documented the important contribution of innate immunity to tumour cell death in women with LLABCs undergoing NAC. The significant associations between the beneficial pathological responses (GPR and pCR) in the tumour microenvironment (primary and ALN metastases) and key innate immune cells (CD163+ TIMs, circulating PMN neutrophils, and DCs/subsets) complement our findings with NK cells and are an important contribution to the understanding of putative anticancer immune responses in NAC, resulting in immune-mediated tumour cell death.
Adriamycin
Axillary lymph node
Cyclophosphamide
Cluster of differentiation
Cytotoxic T lymphocyte
Diaminobenzidine
Disease-free survival
Dendritic cell
Oestrogen receptor
Forkhead box protein 3
Good pathological response
High-power field
Horseradish peroxidase
Indoleamine 2,3-dioxygenase
Immunohistochemistry
Interleukin
Interferon-gamma
Large and locally advanced breast cancer
Monoclonal antibody
Myeloid-derived dendritic cell
Neoadjuvant chemotherapy
Natural killer
Overall survival
Pathological complete response
Plasmacytoid dendritic cell
Polymorphonuclear neutrophil
Poor pathological response
Room temperature
Sentinel lymph node
Suppression of cytokine signalling
Docetaxel
Tumour-associated antigen
T helper
Tumour-infiltrating neutrophil
Tumour-infiltrating macrophage
T regulatory cell
Transforming growth factor-beta
Tumour-infiltrating lymphocyte
Vascular endothelial growth factor
Capecitabine.
The authors declare that they have no competing interests.
Viriya Kaewkangsadan, Chandan Verma, Jennifer M. Eremin, Gerard Cowley, and Oleg Eremin conceptualised and designed the manuscript. Viriya Kaewkangsadan, Chandan Verma, Jennifer M. Eremin, Gerard Cowley, and Oleg Eremin are assigned in the data acquisition. Viriya Kaewkangsadan, Chandan Verma, Jennifer M. Eremin, Gerard Cowley, Mohammad Ilyas, and Oleg Eremin analysed and interpreted the data. Viriya Kaewkangsadan, Chandan Verma, and Gerard Cowley performed the laboratory assays. Viriya Kaewkangsadan, Chandan Verma, Jennifer M. Eremin, and Oleg Eremin wrote the manuscript. Viriya Kaewkangsadan, Chandan Verma, Jennifer M. Eremin, Gerard Cowley, Mohammad Ilyas, Sukchai Satthaporn, and Oleg Eremin reviewed and approved the final version of the manuscript.
The authors wish to acknowledge Mr. Christopher Nolan (Academic Unit of Clinical Oncology, City Hospital, University of Nottingham) for his advice and help with the IHC assays. The clinical trial, from which patients’ tissue specimens and blood samples were collected for the study, was supported by educational grants from Sanofi-Aventis UK, Roche UK, and Chugai UK. The authors wish to acknowledge the financial support provided for this study by a grant from the Nottinghamshire, Derbyshire and Lincolnshire Research Alliance and Candles Charity.