P5-01-04: FOXP3 Positive Regulatory T Lymphocytes and Epithelial FOXP3 Expression in Synchronous Normal, Ductal Carcinoma In Situ and Invasive Cancer of the Breast.
Authors: Lal A , Chan L , DeVries S , Chin K , Benz CC , Chen Y-Y , Hwang S , Waldman FM 
Published: June 2012

Abstract
Background: FOXP3 expressing regulatory T cells (Tregs) have been associated with worse outcome in a number of malignancies, including breast cancer. In this study we evaluate the number of FOXP3 positive T lymphocytes as well as quantitating FOXP3 expression of epithelium in synchronous normal, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) in order to determine whether FOXP3 expression increases with cancer progression.
Materials and Methods: 32 cases containing concurrent normal, DCIS and IDC lesions of the breast were stained for FOXP3 (e-Bioscience) and CD3 (DAKO) using the Envision G/2 Double stain System (DAKO). Multiplex stains were separated by imaging using the CRi's Nuance imager. The ratio of colocalized FOXP3 and CD3 pixels (Treg lymphocytes)/CD3 pixels (all lymphocytes) in adjacent stroma was used as a measure of FOXP3 positive Tregs. Epithelial FOXP3 expression was quantitated by Image J analysis. A subset of the cases (n= 13) were also stained by standard immunohistochemistry for FOXP3. The number of FOXP3 positive lymphocytes/total lymphocytes and the epithelial intensity were scored.
Results: The mean patient age was 52 years. ER, PR and HER2 positivity in the invasive component was 94%, 84% and 16% respectively. One case was triple negative.
The median fraction of FOXP3 lymphocytes increased from normal to DCIS to IDC (0.005, 0.019 and 0.030 respectively). Significant differences were seen for normal vs. IDC (p=&lt;0.0001) and DCIS vs. IDC (p=0.0039). FOXP3 lymphocyte fraction was significantly higher in grade 3 compared to grade 1 tumors (p=0.0140). Standard immunohistochemistry showed similar results (median value for fraction of FOXP3 positive lymphocytes = 0.03, 0.14 and 0.16 in normal, DCIS and IDC respectively). This was significantly different in the normal stroma vs. invasive cancer stroma comparison (p=0.017).
For epithelial FOXP3 staining intensity, the median increased significantly from normal to DCIS (0.130 to 0.175, p=&lt;0.0001) and decreased significantly from DCIS to IDC (0.175 to 0.150, p=0.0181). However, FOXP3 epithelial intensity was higher in grade 3 compared to grade 1 tumors (p=0.0381). Epithelial FOXP3 expression by standard IHC also showed a similar trend (median H score 100, 150 and 120, respectively) with a significant increase in the normal vs. DCIS comparison (p=0.0090).
FOXP3 lymphocyte count had a positive correlation with FOXP3 epithelial intensity in the normal and invasive components (Spearman r=0.4343, p=0.0130 for normal and Spearman r=0.5456, p=0.0012 for IDC).
FOXP3 lymphocyte fraction and FOXP3 epithelial intensity did not vary significantly with size of invasive tumor, nodal status, or stage of disease.
Discussion: FOXP3 is expressed both by tumor infiltrating T lymphocytes as well as tumor epithelium. The significant increase in FOXP3 T regulatory lymphocytes and FOXP3 epithelial expression from normal to invasive cancer suggests a role of FOXP3 in breast carcinogenesis and progression. The significant correlation of FOXP3-T regulatory lymphocytes and FOXP3 epithelial expression in the normal and invasive components suggests expression of one may influence the other early in the breast cancer progression pathway.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-04.