BMC Cancer 2008, 8:57 http://www.biomedcentral.com/1471-2407/8/57 CD3/FOXP3 B7-H1 PD-1 l a m r o N r o m u T A D B E C F IFmigmuurneo1histochemical staining of FOXP3+ Tregs B7-H1 +and PD-1+ in T lymphocytes of breast tissues Immunohistochemical staining of FOXP3+ Tregs B7-H1 +and PD-1+ in T lymphocytes of breast tissues. Repre- sentative micrographs at × 530 magnification of (A&D) CD3/FOXP3 double staining (red color, membranous for CD3 and brown nuclear color for FOXP3 expression). (B&E) B7-H1 single staining (brown color, membranous/cytoplasmic expression). (C&F) PD-1 single staining (brown color, membranous expression). Upper panel (A-C) is sections for normal breast duct and lower panel (D-F) is sections for infiltrating ductal carcinoma of the breast. expression level of FOXP3 in the intratumoral Tregs ranged from weak to intense (Figure 2B). Interestingly, most of FOXP3 intensely stained Tregs were found in close proxim- ity to tumor cells. PD-1+ T lymphocytes were found both intrastromal and intratumoral, where they commonly seen in clumps, or as clumps/single cells respectively. Correlation of FOXP3, B7-H1 and PD-1 expression in TIL with clinicopathological parameters of patients Breast cancer tissues infiltrated with intratumoral FOXP3+ Tregs were found to be significantly associated with patients who had bad prognostic factors such as large tumor size (P = .029), histological grade III (P < .001), estrogen receptor negative status (P = .010) and severe lymphocytic infiltration (P = .051). Intratumoral FOXP3+ Tregs were found also to be associated with progesterone receptor negative status and lymph node metastasis although they did not reach statistical significance (Table 1). The correlation of the expression B7-H1 in tumor cells and FOXP3+ Tregs was also studied. There was a correlation between B7-H1 in tumor cells and FOXP3+ Tregs (P < .019) (Table 1). We have reported previously on the expression of B7-H1 in 50% of 44 breast cancer patients; and found its expres- sion in TIL to be significantly associated with large tumor size, high histological grade III, Her2/neu positive status and severe lymphocytic infiltration [18]. We further exam- ined the B7-H1 expression in TIL of 62 breast cancer patients and correlated its expression with the same prog- nostic factors. Interestingly, very similar correlation was recorded for B7-H1+ TIL, and this was significantly associ- ated with high histological grade III (P = .002), estrogen receptor negative status (P = .029), and sever lymphocytic infiltration (P = .017). There was also a correlation with large tumor size with a borderline significance (P = .072) (Table 1). There was also a correlation between B7-H1 in tumor cells and B7-H1+ TIL (P < .002) (Table 1). Although PD-1 molecule was also expressed by T lym- phocytes infiltrating normal breast tissues lacking B7-H1, its presence in tumor tissues is considered essential for the inhibitory effect of B7-H1 molecule which is abundant in the tumor tissues. Therefore, we tested whether PD-1 is concomitantly expressed in the same patients along with B7-H1+ TIL and FOXP3+ Tregs. We have found that PD-1+ TILs were abundant in patients with high histological grade III (P = .001), estrogen receptor negative status (P = .001) and progesterone receptor negative status (P < .001) (Table 1). There was also a correlation between B7-H1 in tumor cells and PD-1+ TIL (P < .002) (Table 1). Page 4 of 12 (page number not for citation purposes)