BackgroundTumor immune cell infiltrates are essential in hindering cancer progression and may complement the TNM classification. CD8+ and CD163+ cells have prognostic impact in breast cancer but their spatial heterogeneity has not been extensively explored in this type of cancer. Here, their potential as prognostic biomarkers was evaluated, depending on their combined densities in the tumor center (TC) and the tumor invasive margin (IM).MethodsCD8+ and CD163+ cells were quantified by immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples from a cohort totaling 162 patients with histologically-confirmed primary invasive non-metastatic ductal breast cancer diagnosed between 2000 and 2015. Clinical follow-up (median 6.9 years) was available for 97 of these patients.ResultsDifferential densities of CD8+ and CD163+ cells in the combined TC and IM compartments (i.e., high(H)/low(L), respectively for CD8+ cells and the reverse L/H combination for CD163+ cells) were found to have significant prognostic value for survival, and allowed better patient stratification than TNM stage, tumor size, lymph node invasion and histological grade. The combined evaluation of CD8+ and CD163+ cell densities jointly in TC and IM further improves prediction of clinical outcomes based on disease-free and overall survival. Patients having the favorable immune signatures had favorable clinical outcomes despite poor clinicopathological parameters.ConclusionsGiven the important roles of CD8+ and CD163+ cells in regulating opposing immune circuits, adding an assessment of their differential densities to the prognostic biomarker armamentarium in breast cancer would be valuable. Larger validation studies are necessary to confirm these findings.Trial registrationsStudy code: IRB-ID 6079/448/10-6-13Date of approval: 10/06/2013Retrospective study (2000–2010)First patient prospectively enrolled 14/2/2014Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0240-7) contains supplementary material, which is available to authorized users.

Novel molecular markers that address the heterogeneity of breast cancer (BC) and provide meaningful prognostic information for BC patients are needed. Kallikrein-related peptidase 6 (KLK6) is aberrantly expressed and functionally implicated in BC and, like other members of the KLK family, may prove a useful molecular tool for clinical management. Our objective was to assess, for the first time, the clinical relevance of KLK6 mRNA expression in BC. Total RNA was isolated from 165 breast tumors, as well as 100 adjacent non-cancerous tumor specimens. After cDNA synthesis, and following quality control, quantitative real-time PCR for KLK6 expression analysis took place. Receiver operating characteristic curves were constructed in order to assess the ability of KLK6 mRNA expression levels to differentiate between molecular BC subtypes. Survival analyses, using DFS as endpoint, were performed at the univariate and multivariate levels. Publicly available BC databases and online survival analysis tools were used to validate our findings. A significant downregulation of KLK6 mRNA expression was observed in BC tissue sections compared to the non-cancerous component (P < 0.001). The expression of KLK6 is positively associated with tumor grade (P = 0.038) and is overexpressed in TNBC and HER2-positive tumors (P < 0.001). Aberrant KLK6 expression predicts the clinical outcome of BC patients in terms of DFS, independently of currently used prognostic markers (HR = 7.11, 95% CI = 1.19-42.45). The differential expression of KLK6 and its association with unfavorable outcome in BC patients was validated via in silico analyses. Although an independent external cohort is necessary to confirm our findings, we proved for the first time that KLK6 can provide independent prognostic information for BC patients.

This study therefore defines composite immune profiles that identify patients responding to TAAs which may allow better personalisation of cancer therapies.

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It is becoming increasingly clear that the immune profile of breast cancer patients is informative for patient prognosis. Specifically, immune cells with suppressor or effector function have been shown to be valuable in the prediction of patient course on an individual basis. Our previous study has shown that patients with in vitro HER2-reactive peripheral CD8+ T cells, high levels of circulating plasmacytoid dendritic cells (pDCs) but low ratios of Myeloid-Derived Suppressor Cells (MDSCs):pDCs and Regulatory T cells (Tregs):pDCs experience a survival advantage. In the present study we have tested T cell responses to additional tumour associated antigens along with an expanded panel of leukocyte phenotypes including myeloid cells (9 different phenotypes including monocytes, MDSCs, pDCs and monocyte-derived dendritic cells (mDCs)), T cells (5 phenotypes including Tregs) and NK cells in a cohort of 50 prospectively enrolled breast cancer patients with early stage invasive ductal carcinoma. Additionally, we have performed an in vitro expansion of T cells responding to peptides derived from the tumourassociated antigens Survivin and MUC-1, as well as HER-2 to validate our previous results, and influenza peptides as a positive biological control. To characterise the type of the T cell response to these antigens, responding CD4+ and CD8+ cells were assessed for IFNg, TNF, IL-2, IL-5, IL-10 and IL-17 production per cell by intracytoplasmic flow cytometry, producing 6-cytokine T cell polyfunctionality data. Thus, this study sought to assess the relationship between the distribution of peripheral blood myeloid cells, T cells and NK cells and the presence or absence of in vitro T cell responses to tumour-associated antigens in breast cancer. Ongoing analyses will reveal associations between the levels of different leukocyte populations with T cell responses to tumour-associated antigens. Subsequent clinical follow-up will prospectively reveal associations of these factors with patient clinical course.