Purpose: To investigate the relationship of hypoxia-inducible factor-1a (HIF-1a) tumor expression in predicting the response to epirubicin and disease-free survival (DFS) in patients with breast cancer enrolled in a single institution trial of primary anthracycline and tamoxifen therapy. Experimental Design: The expression of HIF-1a was assessed by immunohistochemistry in 187 patients with T 2-4 N 0-1 breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin + tamoxifen as primary systemic treatment. All patients postoperatively received four cycles of the four weekly i.v. CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil). Patients with estrogen receptor (ER)-positive primary tumors also underwent 5 years of treatment with adjuvant tamoxifen. Carbonic anhydrase IX (CAIX) was also scored as a marker of HIF activity. Results: Overall response to therapy progressively decreased with increasing tumor HIF-1a (P < 0.05), and HIF-1awas an independent predictor of response (P < 0.048). HIF-1aexpression was also associated with a significantly shorter DFS (P < 0.02) in all patients and in ER-positive but not in ER-negative patients. Furthermore, CAIX positivity conferred a significantly shorter DFS (P = 0.02) compared with CAIX-negative tumors in patients with HIF-1a-negative tumors. Conclusions: HIF-1a expression in patients with breast cancer is a marker of poor therapy response and outcome, especially in ER-positive patients. The combination of two hypoxia markers has greater utility than assessing just one, and patients with hypoxia markers in their tumors may be suitable for administration of drugs that reduce HIF-1a expression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies.Tumor growth and metastasis is dependent on the generation of a neovasculature. However, newly formed vessels function poorly in supplying oxygen and nutrient requirements in many tumors. Hypoxia, the pathophysiologic consequence of the structurally and functionally disturbed microcirculation (1), is therefore a common feature in solid tumors. Tumors respond to cellular oxygen deprivation using the ubiquitous family of transcription factors known as hypoxia-inducible factors (HIF; ref. 2). Under normal oxygen tension, HIF-1a is hydroxylated by specific prolyl hydroxylases, leading to recognition and binding by the von Hippel-Lindau protein, and targeting for degradation through the proteasome. In conditions of hypoxia, molecular oxygen is not available for hydroxylase activity, which leads to HIF-1a protein stabilization and translocation to the nucleus where it binds to aryl hydrocarbon nuclear translocators resulting in the activation of several gene pathways involved in angiogenesis, glycolysis, erythropoiesis, and apoptosis (see ref.3). Overexpression of HIF-1a protein has been identified in many tumor types (4), with high levels influencing the growth rate and metastatic potential of these cancers. In breast cancers, the frequen...

A B S T R A C T PurposeTo investigate the activity of letrozole plus/minus oral metronomic cyclophophamide as primary systemic treatment (PST) in elderly breast cancer patients. MethodsOne hundred fourteen consecutive elderly women with T2-4 N0-1 and estrogen receptorpositive breast cancer were randomly assigned to primary letrozole therapy (2.5 mg daily for 6 months) or a combination of letrozole plus oral cyclophosphamide (50 mg/daily for 6 months) in an open-labeled, randomized phase II trial. Tumor response was assessed clinically, and tumor Ki67 index and vascular endothelial growth factor (VEGF) -A levels were measured before and after treatment. ResultsOverall response rate was 71.9% (95% CI, 60.0 to 83.8) in the 57 patients randomly assigned to receive primary letrozole and 87.7% (95% CI, 78.6 to 96.2) in the 57 patients randomly assigned to receive letrozole plus cyclophosphamide. The difference in activity between treatment arms was predominantly confined to patients with ductal histology. There was a significantly greater suppression of Ki67 and VEGF-A expression in the letrozole/cyclophosphamide-treated group than in the letrozole-treated group, leading to lower Ki67 and VEGF expression at post-treatment residual histology (P ϭ .03 and P ϭ .002, respectively). ConclusionBoth letrozole and letrozole plus cyclophosphamide treatments appeared active as PST in elderly breast cancer patients. Metronomic scheduling of cyclophosphamide may have an antiangiogenetic effect and the combination of letrozole plus cyclophosphamide warrants testing in a randomized phase III trial.

Purpose: We have shown previously that tumor infiltration by FOXP3 + regulatoryTcells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulatesTreg numbers in mice and promotes the proliferation of humanTregs, we hypothesized that blocking estrogen receptor-a signaling would abrogateTregs and be associated with response to hormonal therapy and increased survival. Experimental Design: FOXP3 + Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T 2-4 N 0-1 ) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral ''metronomic''cyclophosphamide (50 mg/d). Results: Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozolecyclophosphamide patients (P < 0.0001and P < 0.002, respectively) after treatment.Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greaterTreg reduction was observed in responding patients (P < 0.03).Conclusion:This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducingTregs in breast tumors and may be of use in estrogen receptora-negative tumors in combination with immunotherapy approaches.

Activated ERalpha form was an independent factor for sensitivity to chemoendocrine treatment, whereas HIF-1alpha and p44/42 MAPK were independent factors for resistance. Although further confirmatory analyses are needed, these findings have clear potential implications for future strategies in the management of clinical trials with aromatase inhibitors in the breast cancer.