Toll-like receptor-9 expression is inversely correlated with estrogen receptor status in breast cancer

Abstract

Toll-like receptor 9 (TLR9) recognizes microbial and vertebrate DNA. We previously demonstrated TLR9 expression in human breast cancer cell lines and showed that TLR9 ligands stimulate their in vitro invasion. The aim of this study was to characterize TLR9 expression in clinical breast cancer specimens. Immunohistochemical staining intensity was compared with known baseline prognostic factors and distant metastasis-free survival. TLR9 expression was detected in 98% of the tumors studied (n = 141). The mean TLR9 staining intensity was higher in ER- than in the highly ER+ breast cancers (p = 0.039). High-grade tumors had significantly increased TLR9 expression (p = 0.027) compared with lower-grade tumors. The highest TLR9 expression was detected in the mucinous and the lowest in the tubular breast cancers (p = 0.034). Distant metastasis-free survival was higher in the lower TLR9-expressing half of the cohort than in the higher TLR9-expressing group (p = 0.118). TLR9 expression did not correlate with menopausal, PgR or Her2 status, patient age, tumor proliferative or invasive characteristics.

Fig. 1.a

Characterization of the anti-TLR9 antibody was done using tissue sections of paraffin-embedded human tonsil. Anti-CD20 and -CD3 antibodies, which detect B and T lymphocytes, respectively, were used as references to compare TLR9 staining localization within the tissue (×100 objective). Accumulation of CD20+ cells was seen in the B-cell follicles, whereas CD3+ cells were mostly seen in the interfollicular T-cell zones. TLR9 staining was detected in lymphocytes in both the follicles and in the interfollicular areas. The TLR9 staining pattern was intracellular, as detected in lymphocytes in the germ center (×1,000, arrows). b Intracellular TLR9 staining also detected lymphocytes within a capillary (small arrows), whereas no staining was detected if IgG was used instead of the primary anti-TLR9 antibody.

Fig. 2

TLR9 is expressed in breast cancer epithelium. Immunohistochemical detection of TLR9 was seen mostly in epithelial tumor cells and not in the stroma cells in the breast cancer specimen (a). Omission of the primary anti-TLR9 antibody and the use of IgG instead resulted in no staining (b). HE staining of the corresponding tumors (c). Shown are staining results representing high (16), intermediate (7) and negative (0) staining scores of three different specimens.

Fig. 3

Expression of TLR9 is significantly higher in ER– cancers than in highly ER+ breast cancers. TLR9 staining intensities within the individual samples were microscopically scored and stratified according to ER (a), PgR (b) and Her2 expression status (c) or according to triple negativity (ER–, PgR–, Her2–; d) and a combination of positive ER, PgR and/or Her2 status of the tumor. The mean TLR9 staining intensity was significantly higher (* p < 0.05) in the ER– tumors than in highly ER+ (ER3+) tumors.

Fig. 4

Kaplan-Meier survival curve for the probability of distant metastasis-free survival (a) and contralateral breast tumor development (b).