The prolyl hydroxylase enzymes are positively associated with hypoxia-inducible factor-1α and vascular endothelial growth factor in human breast cancer and alter in response to primary systemic treatment with epirubicin and tamoxifen.

INTRODUCTION: The purpose of the present study was to investigate the relationship of expression of hypoxia inducible factor (HIF)-1α-modifying enzymes prolyl hydroxylase (PHD)1, PHD2 and PHD3 to response of tumours and survival in breast cancer patients enrolled in a phase II trial of neoadjuvant anthracycline and tamoxifen therapy. METHODS: The expression of PHD1, PHD2 and PHD3 together with HIF-1α and the HIF-inducible genes vascular endothelial cell growth factor (VEGF) and carbonic anhydrase IX were assessed by immunohistochemistry using a tissue microarray approach in 211 patients with T2-4 N0-1 breast cancer enrolled in a randomised trial comparing single-agent epirubicin versus epirubicin and tamoxifen as the primary systemic treatment. RESULTS: PHD1, PHD2 and PHD3 were detected in 47/179 (26.7%), 85/163 (52.2%) and 69/177 (39%) of tumours at baseline. PHD2 and PHD3 expression was moderate/strong whereas PHD1 expression was generally weak. There was a significant positive correlation between HIF-1α and PHD1 (P = 0.002) and PHD3 (P < 0.05) but not PHD2 (P = 0.41). There was a significant positive relationship between VEGF and PHD1 (P < 0.008) and PHD3 (P = 0.001) but not PHD2 (P = 0.09). PHD1, PHD2 and PHD3 expression was significantly increased after epirubicin therapy (all P < 0.000) with no significant difference in PHD changes between the treatment arms. There was no significant difference in response in tumours that expressed PHDs and PHD expression was not associated with survival. CONCLUSIONS: Although expression of the PHDs was not related to response or survival in patients receiving neoadjuvant epirubicin, our data provide the first evidence that these enzymes are upregulated on therapy in breast cancer and that the biological effects independent of HIF make them therapeutic targets.

Anti-angiogenic effect of tamoxifen combined with epirubicin in breast cancer patients.

Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) are the key factors mediating neo-vascularization. They are often coexpressed in breast cancer. Sex steroids may stimulate angiogenesis via the estrogen receptor (ER) pathway. We investigated to compare the effects of the addition of tamoxifen to epirubicin versus epirubicin alone on VEGF and VEGFR2 expression in breast cancer patients. The expression of VEGF and VEGFR2 was assessed on tissue microarray by immunohistochemistry at baseline conditions and after treatments in the case of 191 patients with T2-4 N0-1 breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin plus tamoxifen as primary systemic treatment. Epirubicin alone failed to induce changes in VEGF expression (P = 0.54), while the addition of tamoxifen to epirubicin resulted in a significant reduction in VEGF expression (P < 0.001). As a consequence, baseline VEGF had a negative prognostic role in patients who received epirubicin alone but not in patients receiving epirubicin plus tamoxifen (interaction test P < 0.05). VEGFR2 expression increased at residual tumor histology in both treatment arms, with a lesser extent in patients receiving tamoxifen plus epirubicin. Decrease in VEGFR2 expression was significantly associated with response rate (P = 0.02). The addition of tamoxifen to epirubicin resulted in a suppression of a key angiogenic pathway. These data suggest a potential synergism of these two drugs.

Immunomodulation of FOXP3+ regulatory T cells by the aromatase inhibitor letrozole in breast cancer patients.

PURPOSE: We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-alpha signaling would abrogate Tregs 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 (T2-4 N0-1) 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 letrozole-cyclophosphamide patients (P < 0.0001 and 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 greater Treg 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 reducing Tregs in breast tumors and may be of use in estrogen receptor-alpha-negative tumors in combination with immunotherapy approaches.

Down-regulation of phosphatidylinositol 3'-kinase/AKT/molecular target of rapamycin metabolic pathway by primary letrozole-based therapy in human breast cancer.

PURPOSE: The phosphatidylinositol 3'-kinase (PI3K)/AKT/molecular target of rapamycin (mTOR) pathway is involved in the development of tumor resistance to endocrine therapy in breast cancer cell lines and represents an attractive target for pharmacologic intervention. However, the effects of endocrine therapy with aromatase inhibitors on in vivo expression of this signaling cascade, and its relation to tumor response and patient outcome, is unknown. EXPERIMENTAL DESIGN: PI3K, phospho-AKT (pAKT) and phospho-mTOR were assessed by immunohistochemistry on tumor specimens collected at baseline and after 6 months of treatment in 113 elderly breast cancer patients consecutively enrolled in a randomized phase II trial of primary letrozole therapy and letrozole associated with metronomic cyclophosphamide. RESULTS: Basal expression of the pathway was not significantly correlated with response or patient outcome. Both letrozole alone and letrozole with cyclophosphamide resulted in a significant reduction of PI3K expression (P = 0.02 and P < 0.005, respectively) and phospho-mTOR expression (P = 0.0001 and P = 0.0001, respectively). pAKT showed no change in the letrozole arm, whereas it was significantly decreased in the letrozole plus cyclophosphamide arm (P < 0.005). pAKT expression reduction was associated with a greater response rate (P = 0.05) and greater reduction in Ki67 expression (P = 0.05). Phospho-mTOR expression reduction was associated with a significantly longer disease-free survival in a multivariate analysis (P = 0.02). CONCLUSIONS: Letrozole inhibits key molecules in the PI3K pathway that are important targets of new drugs being developed to overcome resistance. Changes in these molecules may have prognostic significance. These results should be taken into account when planning prospective trials testing up-front aromatase inhibitor with drugs targeting the PI3K/AKT/mTOR signaling pathway.

Regulation of hepatocyte growth factor activator inhibitor 2 by hypoxia in breast cancer.

PURPOSE: To examine the in vitro regulation of hepatocyte growth factor activator inhibitor type 2 (HAI-2) in breast cancer cells and the in vivo predictive role for the efficacy of chemoendocrine primary therapy in patients with breast cancer. MATERIALS AND METHODS: HAI-2 regulation was studied in a panel of breast cancer cell lines comparing normoxia to hypoxia. The effect of HIF-1alpha RNAi on HAI-2 expression was evaluated in these cells. HAI-2 was examined in breast cancer using in situ hybridization and immunohistochemistry. The HAI-2 predictive role was assessed in T(2-4) N(0-1) breast cancers (n = 177) enrolled in a neoadjuvant randomized trial comparing epirubicin versus epirubicin + tamoxifen. RESULTS: HAI-2 mRNA and protein were regulated by hypoxia in the c-erbB2-positive cell lines, SKBR3 and BT474, and controlled by HIF-1alpha in these cells. Immunohistochemistry confirmed this profile with high expression of HAI-2 in c-erbB2-positive breast cancer. HAI-2 was correlated with T status (P < 0.004), node involvement (P = 0.01), and c-erbB2 expression (P = 0.05). HAI-2 also correlated with hypoxia markers such as carbonic anhydrase IX expression (P = 0.01) and HIF-1alpha. Additionally, high levels of HAI-2 were a significant predictor for poor clinical complete response to preoperative epirubicin in univariate (P = 0.01) and multivariate analyses (P = 0.016). No correlation with disease-free survival and survival was observed. CONCLUSION: HAI-2 expression in breast cancer correlated with tumor aggressiveness in vivo. It is a HIF target in c-erbB2-positive cells and it is an independent negative predictive factor of efficacy of anthracycline therapy. The interaction of HAI-2 with the hepatocyte growth factor activation pathway may be a useful site for therapeutic intervention.

The circadian rhythm of biochemical markers of bone resorption is normally synchronized in breast cancer patients with bone lytic metastases independently of tumor load.

BACKGROUND: Bone metastases are devastating events resulting in disruption of local bone remodeling processes. Physiological bone turnover has a circadian rhythm. No data are available on the circadian pattern of bone turnover markers in patients with bone metastases. METHODS: Twenty post-menopausal women with breast cancer (BC) at first disease relapse and at least one bone metastasis were consecutively recruited. Twenty healthy women served as controls. Patients were free from concomitant chemotherapy/endocrine therapy. Throughout a 24-h period, urine samples were collected at 4-h intervals, and blood samples were collected at 4-h intervals between 08:00 and 24:00, and at 2-h intervals between 24:00 and 08:00. Serum osteocalcin (OC), total and bone-alkaline phosphatase (tALP and bALP, respectively) and C-terminal telopeptide of type I collagen (CTX), and urinary NTX and free deoxypyridinoline (fDPD) were measured together with serum parathyroid hormone (PTH) and serum and urinary calcium and phosphorus. Temporal variations of measured analytes were assessed by ANOVA and the COSINOR model. RESULTS: At 08:00, patients had higher levels of bone resorption indices (NTX, CTX and fDPD) than controls (p<0.0001). tALP and bALP, but not OC, were higher in patients than controls (p<0.001). PTH, serum and urinary calcium and urinary phosphorus did not differ between groups; serum phosphorus was higher in controls (p<0.0001). A circadian rhythm was evident for CTX and fDPD values in both patients and controls. A circadian rhythm in NTX, OC, phosphorus and PTH was apparent in controls only. However, it was detected also in patients when percent changes from MESOR were considered. Serum phosphorus showed a circadian rhythm, while no rhythm was detected for tALP, bALP, serum and urinary calcium. The rhythmicities in cancer patients were normally synchronized, and rhythmic parameters were independent of tumor load in the skeleton, age and menopausal status. CONCLUSIONS: This is the first study to yield information on the maintenance of the temporal program of bone turnover in bone metastatic cancer patients. Whether administration of bisphosphonates in the nighttime leads to a different outcome with respect to the current administration in the morning is a matter of future research.

Hypoxia-inducible factor-1alpha expression predicts a poor response to primary chemoendocrine therapy and disease-free survival in primary human breast cancer.

PURPOSE: To investigate the relationship of hypoxia-inducible factor-1alpha (HIF-1alpha) 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-1alpha 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-1alpha (P < 0.05), and HIF-1alpha was an independent predictor of response (P < 0.048). HIF-1alpha expression 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-1alpha-negative tumors. CONCLUSIONS: HIF-1alpha 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-1alpha expression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies.

Hypoxia-related biological markers as predictors of epirubicin-based treatment responsiveness and resistance in locally advanced breast cancer.

PURPOSE: To identify hypoxia-related biomarkers indicative of response and resistance to epirubicin treatment in patients with locally advanced breast cancer. PATIENTS AND METHODS: One hundred seventy-six women with T2-4 N0-1 breast tumours were randomly assigned to receive epirubicin 120 mg/m2/1-21 (EPI ARM), epirubicin 120 mg/m2/1-21 + erythropoietin 10.000 IU sc three times weekly (EPI-EPO ARM) and epirubicin 40 mg/m2/w-q21 (EPI-W ARM). Sixteen tumour proteins involved in cell survival, hypoxia, angiogenesis and growth factor, were assessed by immunohistochemistry in pre-treatment samples. A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization. RESULTS: VEGF and GLUT-1 expression were significantly positively associated with complete response (CR) to treatment in all leave-one-out iterations. Bcl-2 expression was inversely correlated with pCR, whilst EPO expression was positively correlated with pathological complete response (pCR). Haemaglobin and HIF-1a nuclear expression were inversely correlated with pCR. HB and HIF-1a expression were associated with a higher risk of relapse and overall survival. CONCLUSION: Hypoxic biomarkers determines the epirubicin resistance in breast cancer. Assessment of such biomarkers, may be useful for predicting chemosensitivity and also anthracycline-based treatment outcome.

Role of carbonic anhydrase IX expression in prediction of the efficacy and outcome of primary epirubicin/tamoxifen therapy for breast cancer.

The purpose of this study is to investigate the role of carbonic anhydrase IX (CAIX) expression in predicting the response to epirubicin and disease-free survival (DFS) in breast cancer patients enrolled in a single institution trial of primary anthracycline and tamoxifen therapy. CAIX expression was assessed in 183 patients with T2-4 N0-1 breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin+tamoxifen as primary systemic treatment. All patients received postoperatively four cycles of the four weekly i.v. cyclophosphamide, methotrexate, 5-fluorouracil regimen. Patients with estrogen receptor (ER)-positive primary tumors received 5 years of adjuvant tamoxifen. Pretreatment, p53 (P=0.007), c-erbB2 (P<0.01), and Ki67 (P=0.02) were directly associated with CAIX expression, while bcl2 (P<0.000) and ER (P=0.000) and progesterone receptor (PgR; P<0.01) were inversely correlated. In multivariate analysis, only high p53 and low bcl2 were independently associated with CAIX positivity. CAIX immunostaining was significantly associated with poor outcome for DFS (P<0.002) and overall survival (P=0.001). In multivariate analysis, a significant interaction was found between CAIX and markers of hormone sensitivity, bcl2 (P=0.01), ER (P=0.02), PgR (P=0.02), and lymph node involvement (P=0.04), in predicting DFS. Presently, there are few clinical markers of resistance to tamoxifen treatment in ER-positive tumors. CAIX expression in breast cancer patients shows a negative predictive role of treatment efficacy in ER-positive patients on the adjuvant tamoxifen after primary chemo-endocrine therapy. Studies investigating the effects of pH on tamoxifen uptake and the effects of therapy with CA inhibitors are planned.

Combination regimen of epirubicin, vinorelbine and 5-fluorouracil continuous infusion as first-line chemotherapy in anthracycline-naive metastatic breast cancer patients.

We investigated the activity and toxicity of a combination of vinorelbine 25 mg/m2 on days 1 and 15; epirubicin 25 mg/m2 on days 1, 8, 15; and 5-fluorouracil continuous infusion at 200 mg/m2 every day, administered as first-line chemotherapy in anthracycline-naive metastatic breast cancer patients. Fifty-three patients entered the study. Cycles were repeated every 28 days. Objective response was 60% by World Health Organisation (WHO) criteria and 63% by Response Evaluation Criteria in Solid Tumours (RECIST). The median time to progression was 12.7 months (17.6 months in responders) and the median survival duration was 32.9 months. The dose-limiting toxicity was leucopenia (grade 3/4 in 36% of patients). Grade 3/4 non-haematological toxicities included mucositis in 11% of patients, skin and cardiac toxicity in 4% and 2%, respectively. The combination of vinorelbine, epirubicin and 5-fluorouracil continuous infusion was found to be an active and manageable first-line regimen for metastatic breast cancer patients.

Phosphorylated ERalpha, HIF-1alpha, and MAPK signaling as predictors of primary endocrine treatment response and resistance in patients with breast cancer.

PURPOSE: We aimed to identify signaling pathways involved in the response and resistance to aromatase inhibitor therapy in patients with breast cancer. PATIENTS AND METHODS: One hundred fourteen women with T2-4 N0-1, estrogen receptor (ER) alpha-positive tumors were randomly assigned to neoadjuvant letrozole or letrozole plus metronomic cyclophosphamide. Twenty-four tumor proteins involved in apoptosis, cell survival, hypoxia, angiogenesis, growth factor, and hormone signaling were assessed by immunohistochemistry in pretreatment samples (eg, caspase 3, phospho- mammalian target of rapamycin, hypoxia-inducible factor 1alpha [HIF-1alpha], vascular endothelial growth factor, mitogen-activated protein kinase [MAPK], phosphorylated epidermal growth factor receptor, phosphorylated ERalpha [pERalpha]). A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization. Ten-fold cross-validation and iterative leave-one-out were employed to validate and test the model, respectively. Tumor size, nodal status, age, tumor grade, histological type, and treatment were included in the analysis. RESULTS: Ninety-one patients (81%) attained a disease response, 48 achieved a complete clinical response (43%) whereas 22 did not respond (19%). Increased pERalpha and decreased p44/42 MAPK were significant factors for complete response to treatment in all leave-one-out iterations. Increased p44/42 MAPK and HIF-1alpha were significant factors for treatment resistance in all leave-one-out iterations. There was no significant interaction between these variables and treatment. CONCLUSION: 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.

Randomized phase II trial of letrozole and letrozole plus low-dose metronomic oral cyclophosphamide as primary systemic treatment in elderly breast cancer patients.

PURPOSE: To investigate the activity of letrozole plus/minus oral metronomic cyclophosphamide as primary systemic treatment (PST) in elderly breast cancer patients. METHODS: One hundred fourteen consecutive elderly women with T2-4 N0-1 and estrogen receptor-positive 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. RESULTS: Overall 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 = .03 and P = .002, respectively). CONCLUSION: Both letrozole and letrozole plus cyclophosphamide treatments appeared active as PST in elderly breast cancer patients. Metronomic scheduling of cyclophosphamide may have an antiangiogenic effect and the combination of letrozole plus cyclophosphamide warrants testing in a randomized phase III trial.