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. 2012 Apr 3:12:134.
doi: 10.1186/1471-2407-12-134.

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

Affiliations

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

Raoul Droeser et al. BMC Cancer. .

Abstract

Background: Clinical relevance of tumor infiltrating lymphocytes (TILs) in breast cancer is controversial. Here, we used a tumor microarray including a large series of ductal and lobular breast cancers with long term follow up data, to analyze clinical impact of TIL expressing specific phenotypes and distribution of TILs within different tumor compartments and in different histological subtypes.

Methods: A tissue microarray (TMA) including 894 ductal and 164 lobular breast cancers was stained with antibodies recognizing CD4, FOXP3, and IL-17 by standard immunohistochemical techniques. Lymphocyte counts were correlated with clinico-pathological parameters and survival.

Results: CD4(+) lymphocytes were more prevalent than FOXP3(+) TILs whereas IL-17(+) TILs were rare. Increased numbers of total CD4(+) and FOXP3(+) TIL were observed in ductal, as compared with lobular carcinomas. High grade (G3) and estrogen receptor (ER) negative ductal carcinomas displayed significantly (p < 0.001) higher CD4(+) and FOXP3(+) lymphocyte infiltration while her2/neu over-expression in ductal carcinomas was significantly (p < 0.001) associated with higher FOXP3(+) TIL counts. In contrast, lymphocyte infiltration was not linked to any clinico-pathological parameters in lobular cancers. In univariate but not in multivariate analysis CD4(+) infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3(+)/CD4(+) ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033).

Conclusions: Ductal and lobular breast cancers appear to be infiltrated by different lymphocyte subpopulations. In ductal cancers increased CD4(+) and FOXP3(+) TIL numbers are associated with more aggressive tumor features. In survival analysis, absolute numbers of TILs do not represent major prognostic indicators in ductal and lobular breast cancer. Remarkably however, a ratio > 1 of total FOXP3(+)/CD4(+) TILs in ductal carcinoma appears to represent an independent favorable prognostic factor.

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Figures

Figure 1
Figure 1
Examples of CD4+, FOXP3+ and IL-17+ infiltrating lymphocytes in ductal and lobular breast cancer. A, B, C: Immunohistochemical staining of breast cancer tissue punches incubated with anti-CD4 antibody (brown staining): A: Ductal breast cancer with predominantly stromal and intratumoral CD4+ cells. B: Ductal breast cancer with prominent cell pleomorphism and easily detectable intratumoral CD4+ cells, within the tumor and surrounding it. C: Example of an invasive lobular breast cancer with CD4+ cells. D; E: Zoom of breast cancers incubated with anti-FOXP3 antibody (brown staining): D: Invasive ductal breast cancer with clear intratumoral infiltration by FOXP3+ cells. E: Invasive lobular breast cancer with stromal and intratumoral FOXP3+ cells. F: Ductal breast cancer showing a single infiltrating IL-17+ cell (brown staining). G/H: Ductal carcinoma with a FOXP3/CD4 ratio > 1. G shows the FOXP3 staining and H the CD4 staining. I/J: Lobular cancer with a FOXP3/CD4 ratio < 1. I shows the FOXP3 staining and J the CD4 staining.
Figure 2
Figure 2
Examples of CD3 and CD163 specific staining. Immunohistochemistry of invasive ductal breast cancer infiltrated by CD3+ (A, C) and CD163+ (B, D) cells (brown staining) in tumor samples with FOXP3/CD4 ratios > 1 (A, B) and < 1 (C, D).
Figure 3
Figure 3
Box plot analysis of CD4+, FOXP3+ and IL-17+ cell distribution in ductal versus lobular carcinoma. Box plot analysis of lymphocyte infiltration in ductal versus lobular carcinoma for CD4+, FOXP3+ and IL-17+ TILs. After adjustment for multiple comparisons, only p < 0.001 was considered statistically significant.
Figure 4
Figure 4
Overall survival analysis in patients with ductal breast cancer. A: Overall survival in patients with ductal carcinomas and high and low level of CD4+ TILs. Kaplan-Meier survival curves (univariate-analysis) of ductal breast cancers and overall survival in patients with high level of CD4+ versus low level of CD4+ cells infiltrating the tumor. CD4+ infiltrating cells: high level of CD4+ violet curve; low level of CD4+ black curve. Patients with high level of CD4+ cell infiltration show a significant (p = 0.031) worse survival (cut-off established by ROC curves: > 6 CD4+ TILs). B: Overall survival in patients with ductal carcinomas according to total FOXP3/CD4 ratio > or < 1. Kaplan-Meier survival curves (univariate-analysis) of ductal breast cancers and overall survival in patients with ratio of total FOXP3+/CD4+ cells > 1 (blue curve) or < 1 (black curve). Patients with a > 1 ratio had a significantly better survival (p = 0.0005).

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