Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) have garnered an extraordinary amount of interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs induce type I interferons and other cytokines, which drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, 4 and 9 in breast cancer.</p> <p>Methods</p> <p>The expression levels of TLR3, TLR4 and TLR9 were analyzed on tumors from 74 patients with breast cancer. The analysis was performed by immunohistochemistry.</p> <p>Results</p> <p>Samples of carcinomas with recurrence exhibited a significant increase in the mRNA levels of TLR3, TLR4 and TLR9. Tumors showed high expression of TLRs expression levels by cancer cells, especially TLR4 and 9. Nevertheless, a significant percentage of tumors also showed TLR4 expression by mononuclear inflammatory cells (21.6%) and TLR9 expression by fibroblast-like cells (57.5%). Tumors with high TLR3 expression by tumor cell or with high TLR4 expression by mononuclear inflammatory cells were significantly associated with higher probability of metastasis. However, tumours with high TLR9 expression by fibroblast-like cells were associated with low probability of metastasis.</p> <p>Conclusions</p> <p>The expression levels of TLR3, TLR4 and TLR9 have clinical interest as indicators of tumor aggressiveness in breast cancer. TLRs may represent therapeutic targets in breast cancer.</p

Impact of CD68/(CD3+CD20) ratio at the invasive front of primary tumors on distant metastasis development in breast cancer.

Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68⁺), T-cells (CD3⁺ and B-cells (CD20⁺) at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs) and their inhibitors (TIMPs) either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20) ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2). In addition, a high CD68/(CD3+CD20) ratio (>0.05) was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20) ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23-5.24), p<0.01). Therefore, CD68/(CD3+CD20) ratio at the invasive front could be used as an important prognostic marker

Cox's univariate (HR) and multivariate (RR) analysis of the significant relationships between MMPs, TIMPs expression or CD68/(CD3+CD20) ratio at the tumor center or at the invasive front, and relapse-free survival.

<p>Abbreviations: MIC: mononuclear inflammatory cells; HR: hazard ratio; RR: relative risk; CI: confidence interval.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.005;</p>****<p>p<0.001.</p

Relationship between inflammatory cells count or ratio and clinico- pathological characteristics in 102 patients with invasive ductal carcinoma of the breast.

<p>Mann-Whithney or Kruskall-Wallis tests.</p

Distribution of the total number of CD markers by mm² at the invasive front, in 102 breast carcinomas.

<p>CD3 (A), CD20 (B) and CD68 (C).</p

Basal characteristics of 102 patients with invasive ductal carcinoma of the breast.

<p>Basal characteristics of 102 patients with invasive ductal carcinoma of the breast.</p

Representative example of immunostaining.

<p>MMP11 (A) and TIMP2 (B) immunostaining at the tumor center and MMP9 (C) and MMP14 (D) at the invasive front (×200 magnification), indicating the different cell types. Tumor cells (★), lymphocytes (<>\raster(70%)="rg2"<>) and macrophages (<>\raster(70%)="rg1"<>).</p

Relationship between inflammatory cells count or ratio at invasive front and MMPs/TIMPs expression by mononuclear inflammatory cells at invasive front or tumor center.

<p>Mann-Whithney test. MICs: mononuclear inflammatory cells. Data are expressed as median (range). N.S: not significant.</p