BMC Cancer 2008, 8:57 http://www.biomedcentral.com/1471-2407/8/57 Table 2: Correlation between combined FOXP3+ T-reg and B7- H1 with the clinicopathological parameters of 62 breast cancer patients FOXP3 and B7-H1 Expression Both + One + Both - Age < 40 years ≥ 40 years Tumor Size <4 cm ≥ 4 cm Lymph Node Metastasis Negative 1 to 3 4 to 9 10 or more Histological Grade (SBR) II III Lymphovascular Invasion Positive Negative Her2/neu Status Positive Negative ER Status Positive Negative PR Status Positive Negative Neo-adjuvant Chemotherapy With Without Lymphocyte Infiltration Focal Moderate Severe 21 (55)♣ 10 (45) 18 (64) 13 (41) 12 (63) 4 (33) 9 (50) 5 (56) 9 (30) 22 (73) 20 (56) 11 (46) 16 (57) 15 (47) 16 (40) 15 (75) 11 (38) 20 (65) 17 (49) 14 (56) 10 (38) 9 (47) 12 (80) Abbreviations: (+ and -) are number of positive and negative patients, ♣Numbers between brackets are the percentages of patients, Two sample had unknown LN+ status, *P values in bold represent a significant data. abundant in tumor tissues of 56% of breast cancer patients but absent in normal tissues adjacent to the tumors. FOXP3+ Tregs infiltrating tumors have been 7 (18) 7 (32) 6 (21) 8 (25) 3 (16) 3 (25) 5 (28) 3 (33) 7 (23) 7 (23) 8 (22) 6 (25) 7 (25) 7 (22) 7 (24) 7 (23) 9 (26) 5 (20) 5 (19) 7 (37) 2 (13) 10 (25) 4 (20) 14 (35) 0.017 1 (5) 11 (42) 0.022 3 (16) 1 (7) *P 0.496 0.128 0.605 0.745 0.487 0.054 0.828 14 (47) <0.001 1 (3) 10 (26) 5 (23) 4 (14) 11 (34) 4 (21) 5 (42) 4 (22) 1 (11) 8 (2) 7 (29) 5 (18) 10 (31) 11 (38) 4 (13) 9 (26) 6 (24) reported by other studies [25,26,38]. Abundant accumu- lation of Tregs in tumor tissues might be due to the induc- tion of CD4+CD25+ Tregs from peripheral CD4+CD25- T lymphocytes [39,40] and/or migration of Tregs from other parts of the body to the tumor area by chemotactic factors like CCL22 [24,41]. This hypothesis is supported by increase of Tregs in peripheral blood of cancer patients [25,42]. Standard prognostic factors linked to high-risk breast can- cer patients include: young age, large tumor size, high his- tological grade, positive lymph node metastasis and negative hormonal receptors status [43]. We have demon- strated that FOXP3+ Tregs infiltrating tumor tissues corre- lates significantly with important bad prognostic factors: large tumor size (P = .029), high histological grade (P < .001) and estrogen receptor negative status (P = .010). Similar findings have been recently reported by Bates et al showing an association between high Tregs numbers, and high histological grade and estrogen receptor negative sta- tus [26]. Estrogen receptor negative breast tumors are gen- erally associated with poor prognosis which is usually attributed to the association with high proliferative rate and lack of differentiation [44], that are features of pro- genitors of stem-like cells. Indeed it has been shown recently that stem cells obtained from both normal and cancer breast tissues lack the estrogen receptor [45]. Such stem cells were found to produce TGFβ1 [45] which is known to induce Tregs [46]. The association of FOXP3+ Tregs with bad prognostic factors may suggest a contribution of Tregs infiltrating breast cancer tissues to tumor escape from the immune system as their depletion lead to tumor rejec- tion in animal models [47]. We have also confirmed the B7-H1 expression in TIL and its correlation with bad prog- nostic factors reported in our previous study [18]. The association of B7-H1 expression in TIL and FOXP3+ Tregs infiltration with the same bad prognostic factors suggests that both molecules correlate with each other. Indeed, there was a highly significant correlation between the expression of FOXP3+ Tregs and B7-H1 (P < .0001). We have also found that PD-1 expression in TIL correlated with bad prognostic factors. PD-1 has been reported to be expressed in normal tissues to regulate self-reactive T cell responses [48]. In the present study PD-1 was expressed in up to 30% of T lymphocytes from normal breast tissues while up to 80% of TIL were PD-1+. The abundant expres- sion of PD-1 in TIL together with B7-H1 (absent in nor- mal tissues) seen in the present study may contribute to downregulation of the patients' immune response [4,6]. Furthermore, combination of FOXP3+ Tregs, B7-H1+ TIL and PD-1+ TIL were found to be highly associated with patients who had high histological grade III (P < .001) and estrogen receptor negative tumors (P < .007) sugges- tive of synergistic contribution to bad prognosis. It seems Page 8 of 12 (page number not for citation purposes)