PURPOSE Preclinical data suggest a contribution of the immune system to chemotherapy response. In this study, we investigated the prespecified hypothesis that the presence of a lymphocytic infiltrate in cancer tissue predicts the response to neoadjuvant chemotherapy. METHODS We investigated intratumoral and stromal lymphocytes in a total of 1,058 pretherapeutic breast cancer core biopsies from two neoadjuvant anthracycline/taxane-based studies (GeparDuo, n = 218, training cohort; and GeparTrio, n = 840, validation cohort). Molecular parameters of lymphocyte recruitment and activation were evaluated by kinetic polymerase chain reaction in 134 formalin-fixed, paraffin-embedded tumor samples. Results In a multivariate regression analysis including all known predictive clinicopathologic factors, the percentage of intratumoral lymphocytes was a significant independent parameter for pathologic complete response (pCR) in both cohorts (training cohort: P = .012; validation cohort: P = .001). Lymphocyte-predominant breast cancer responded, with pCR rates of 42% (training cohort) and 40% (validation cohort). In contrast, those tumors without any infiltrating lymphocytes had pCR rates of 3% (training cohort) and 7% (validation cohort). The expression of inflammatory marker genes and proteins was linked to the histopathologic infiltrate, and logistic regression showed a significant association of the T-cell-related markers CD3D and CXCL9 with pCR. CONCLUSION The presence of tumor-associated lymphocytes in breast cancer is a new independent predictor of response to anthracycline/taxane neoadjuvant chemotherapy and provides useful information for oncologists to identify a subgroup of patients with a high benefit from this type of chemotherapy.

Tumor-Associated Lymphocytes As an Independent Predictor of Response to Neoadjuvant Chemotherapy in Breast Cancer

The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). Clinically, we found that high numbers of CD163+ M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

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Human breast cancer cells educate macrophages toward the M2 activation status

Tumor-associated macrophages as potential diagnostic and prognostic biomarkers in breast cancer

Background Macrophages constitute a major component of the leucocytic infiltrate of tumours. Human studies show an association between tumour-associated macrophages and tumours with poor prognostic features. In breast cancer, the presence of macrophages has been correlated with increased angiogenesis and poor prognosis but little information is available about the independent prognostic role of macrophages infiltrating breast carcinomas. Aims and methods This study used immunohistochemistry and tissue microarrays to assess the density and localisation of CD68 macrophages infiltrating 1322 breast tumours and to identify any relationship with clinicopathological factors and patient outcome. Results Tumour-infiltrating macrophages were present in the majority of tumours with a predominantly diffuse pattern. The density of distant stromal macrophages (infiltrating stroma away from the carcinoma, median count 14 cells) was higher than intratumoural (median zero cells) and adjacent stromal macrophages (median three cells). Higher total macrophage number was associated with higher tumour grade (r s =0.39, p Conclusions Macrophages are heterogeneous with different subsets having different functions. The present study suggests that overall macrophage numbers are not related to prognosis in breast cancer. However, further studies are needed to investigate the potential role of different subsets of macrophages.

https://jcp.bmj.com/content/jclinpath/65/2/159.full.pdf

Tumour-infiltrating macrophages and clinical outcome in breast cancer

Background The prognostic values of tumor-infiltrating lymphocytes (TILs) and TILs subsets in breast cancer (BC) are uncertain. Methods A systematic literature search (MEDLINE, Web of Science, EMBASE, and the Cochrane Library to August 2014) was conducted for studies which met the eligibility criteria. The primary clinical outcome was defined as disease-free survival (DFS), overall survival (OS), and BC-specific survival (BCSS). Random or fixed-effects model was adopted to estimate the summary hazard ratio (HR). Results Twenty-five published studies comprising 22,964 patients were reviewed. Pooled analysis indicated that TILs were not prognostic markers for DFS and OS in overall population, but related to improved DFS (HR, 0.82; 95% CI, 0.76–0.88) and OS (HR, 0.79; 95% CI, 0.71–0.87) in triple negative breast cancer (TNBC) patients. For TILs subsets, CD8+ lymphocytes were associated with improved DFS (HR, 0.69; 95% CI, 0.56–0.84) and BCSS (HR, 0.78; 95% CI, 0.71–0.86) in overall population, while FOXP3+ lymphocytes were associated with reduced DFS (HR, 1.47; 95% CI, 1.01–2.05) and OS (HR, 1.50; 95% CI, 1.15–1.97). In estrogen receptor (ER) negative patients, CD8+ lymphocytes was also related to better BCSS. In addition, the high density of CD20+, CD3+ or low level of PD-1+ or γδ T lymphocytes indicated increased OS in limited studies. Conclusion TILs and TILs subsets are promising prognostic biomarkers in breast cancer, especially in TNBC.

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0152500&type=printable

The Prognostic Value of Tumor-Infiltrating Lymphocytes in Breast Cancer: A Systematic Review and Meta-Analysis

The significance of the inter-relationship between tumour and host local/systemic inflammatory responses in primary operable invasive breast cancer is limited. The inter-relationship between the systemic inflammatory response (pre-operative white cell count, C-reactive protein and albumin concentrations), standard clinicopathological factors, tumour T-lymphocytic (CD4+ and CD8+) and macrophage (CD68+) infiltration, proliferative (Ki-67) index and microvessel density (CD34+) was examined using immunohistochemistry and slide-counting techniques, and their prognostic values were examined in 168 patients with potentially curative resection of early-stage invasive breast cancer. Increased tumour grade and proliferative activity were associated with greater tumour T-lymphocyte (P<0.05) and macrophage (P<0.05) infiltration and microvessel density (P<0.01). The median follow-up of survivors was 72 months. During this period, 31 patients died; 18 died of their cancer. On univariate analysis, increased lymph-node involvement (P<0.01), negative hormonal receptor (P<0.10), lower albumin concentrations (P<0.01), increased tumour proliferation (P<0.05), increased tumour microvessel density (P<0.05), the extent of locoregional control (P<0.0001) and limited systemic treatment (P⩽0.01) were associated with cancer-specific survival. On multivariate analysis of these significant covariates, albumin (HR 4.77, 95% CI 1.35–16.85, P=0.015), locoregional treatment (HR 3.64, 95% CI 1.04–12.72, P=0.043) and systemic treatment (HR 2.29, 95% CI 1.23–4.27, P=0.009) were significant independent predictors of cancer-specific survival. Among tumour-based inflammatory factors, only tumour microvessel density (P<0.05) was independently associated with poorer cancer-specific survival. The host inflammatory responses are closely associated with poor tumour differentiation, proliferation and malignant disease progression in breast cancer.

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The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic and macrophage infiltration, microvessel density and survival in patients with primary operable breast cancer

J C Doughty

Papers

The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic and macrophage infiltration, microvessel density and survival in patients with primary operable breast cancer