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Supplementary Data from 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-α 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-α-negative tumors in combination with immunotherapy approaches.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading throughout the world. The study describes 12 patients with SARS-CoV-2 pneumonia, who developed an acute erythematous rash with nonfollicular pinhead-sized pustules, without mucosal involvement. The clinical differential diagnosis was viral rash, acute generalized exanthematous pustulosis (AGEP), or multiform erythema. computed tomography with a diagnosis of interstitial pneumonia and a respiratory tract sample positive for SARS-CoV-2 in a reverse transcriptase polymerase chain reaction assay. Patients had signs of respiratory distress and were treated with hydroxychloroquine, darunavir, ritonavir, heparin, ceftriaxone, and azithromycin. Punch biopsies showed subcorneal pustules typical of AGEP. Dermal microvascular injury and thrombosis as described in skin damage by SARS-CoV-2 infection was not observed. The direct immunofluorescence for IgG, IgA, IgM, and C3 was negative in 8 patients investigated. A polymerase chain reaction for RNA SARS-CoV-2 performed on frozen skin was negative in 5 of 6 patients. Most of our patients were treated with systemic corticosteroids. After some days (4-10), the diffuse erythema and pustules had improved. AGEP is classified as a severe cutaneous adverse reaction, provoked by drugs and acute infections. Characteristically, removal of the offending agent leads to spontaneous resolution typically in less than 15 days. The recognition of AGEP is important, in order to avoid confusion with a systemic infection and consequently to avoid incorrect treatment. Cutaneous adverse reactions to drugs are common and are major health problems worldwide causing considerable costs for health care systems. We suggest that in the patients with AGEP during SARS-CoV-2 pneumonia, viral infection is a risk factor for developing drug reaction.

A key tool for monitoring breast cancer patients under neoadjuvant treatment is the identification of reliable predictive markers. Ki67 has been identified as a prognostic and predictive marker in ER-positive breast cancer. Ninety ER-positive, HER2 negative locally advanced breast cancer patients received letrozole (2.5 mg daily) and cyclophosphamide (50 mg daily) with/without Sorafenib (400 mg/bid daily) for 6 months before undergoing surgery. Ki67 expression and tumor size measured with caliber were determined at baseline, after 30 days of treatment and at the end of treatment. Patients were assigned to a clinical response category according to Response Evaluation Criteria in Solid Tumors, both at 30 days and before surgery and further classified as high-responder and low-responder according to the median variation of Ki67 values between biopsy and 30 days and between biopsy and surgery time. The predictive role of Ki67 and its changes with regard to clinical response and survival was analyzed. No differences in terms of survival outcomes emerged between the arms of treatment, while we observed a higher percentage of women with progression or stable disease in arm with the combination containing Sorafenib (20.5% vs 7.1%, p = 0.06). Clinical complete responders experienced a greater overall variation in Ki67 when compared with partial responders and patients with progressive/stable disease (66.7% vs 30.7%, p = 0.009). High responders showed a better outcome than low responders in terms of both disease-free survival ( p = 0.009) and overall survival ( p = 0.002). ΔKi67 score evaluated between basal and residual tumor at definitive surgery showed to be highly predictive of clinical complete response, and a potential parameter to be used for predicting disease-free survival and overall survival in luminal breast cancer treated with neoadjuvant endocrine-based therapy.

INTRODUCTION: Neoadjuvant hormonal therapy is generally considered a valid option for hormone receptor positive breast cancer (BC) patients who are unfit for chemotherapy or surgery. AIMS: Whilst numerous studies analyzed efficacy of neoadjuvant chemotherapy (CT) or endocrine therapy (HT) alone in hormone receptor positive patients, there is a lack of research looking at the usefulness of a preoperative combinatorial approach of CT and HT in this patient subgroup. METHODS: Using a predictive model previously described in the literature, developed to analyze the probability of benefit from preoperative chemotherapy, we were able to compare pathological complete response (pCR) rates expected with the use of CT alone with the pCR rates reported in a population of 192 patients treated with the combination of tamoxifen plus anthracycline-based CT at Cremona Hospital between 2003 and 2006. RESULTS: Even with a relatively small patient population, this approach provided insightful information for the selection of hormone receptor positive BC patients most likely to benefit from the use of preoperative HT and CT in combination. Whilst no statistically significant benefit was obtained with the addition of tamoxifen to neoadjuvant chemotherapy in the entire population, or in any of the molecular stratification subgroups, the analysis of the calibration curve showed that a combinatorial approach may improve pCR in patients with luminal B tumors. More specific trials should be designed to confirm our initial results. CONCLUSION: To the best of our knowledge, this is the first report investigating the efficacy of the combination of CT and HT in the neoadjuvant treatment of hormone receptor positive BC.

Background Endocrine treatment with Tamoxifen and aromatase inhibitors (AIs) is a staple in the management of hormone receptor positive breast cancer (HR + BC). It has become clear that HR + BC carries a consistent risk of relapse up to 15 years post-diagnosis. While increasing evidence supports the use of extended adjuvant Tamoxifen over 5 years, controversial data are available on the optimal duration of extended AIs adjuvant treatment. We performed a meta-analysis to assess the real impact of extended adjuvant therapy with AIs on disease-free survival (DFS). Methods A literature-based meta-analysis of randomized controlled trials (RCTs) was undertaken. Relevant publications from PubMed, the Cochrane Library, and abstracts from American Society of Clinical Oncology (ASCO) and San Antonio Breast Cancer (SABCS) symposia were searched. Primary and secondary endpoints were Disease Free Survival (DFS) and overall survival (OS) respectively. A subgroup analysis was also performed to elucidate the impact of nodal involvement. Results The pooled analysis revealed a significant increase in DFS in the extended AIs group (hazard ratio (HR): 0.78, 95% CI: 0.68–0.90; P = 0.0006). The subgroup analysis according to nodal status showed a greater DFS benefit with extended AIs in patients with positive nodes (HR = 0.67 versus 0.80). Our analysis also demonstrated no improvement in OS with extended AIs (HR = 0.99, 95%CI: 0.87–1.12; P = 0.84). Conclusion This work confirmed the efficacy of extended adjuvant treatment with AIs for HR + early breast cancer, with a 22% increase in DFS, but no impact on OS. Greater efficacy was observed in women with positive nodal status.

Background Histological status of axillary lymph nodes is an important prognostic factor in patients receiving surgery for breast cancer (BC). Sentinel lymph node (SLN) biopsy (B) has rapidly replaced axillary lymph node dissection (ALND), and is now the standard of care for axillary staging in patients with clinically node-negative (N0) operable BC. The aim of this study is to compare pretreatment lymphoscintigraphy with a post primary systemic treatment (PST) scan in order to reduce the false-negative rates for SLNB. Methods In this single-institution study we considered 170 consecutive T2-4 N0-1 M0 BC patients treated with anthracycline-based PST. At the time of incisional biopsy, we performed sentinel lymphatic mapping. After PST, all patients repeated lymphoscintigraphy with the same methodology. During definitive surgery we performed further sentinel lymphatic mapping, SLNB and ALND. Results The SLN was removed in 158/170 patients giving an identification rate of 92.9% (95% confidence interval (CI) = 88.0–96.3%) and a false-negative rate of 14.0% (95% CI = 6.3–25.8%). SLNB revealed a sensitivity of 86.0% (95% CI = 74.2–93.7%), an accuracy of 94.9% (95% CI = 90.3–97.8%) and a negative predictive value of 92.7% (95% CI = 86.1–96.8%). Conclusion Identification rate, sensitivity and accuracy are in accordance with other studies on SLNB after PST, even after clinically negative node conversion following PST. This study confirms that diagnostic biopsy and neoadjuvant chemotherapy maintain breast lymphatic drainage unaltered.

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. © 2011 Fox et al.; licensee BioMed Central Ltd.

PurposeWe aimed to identify signaling pathways involved in the response and resistance to aromatase inhibitor therapy in patients with breast cancer.Patients and MethodsOne hundred fourteen women with T2-4 N0-1, estrogen receptor (ER) α–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 1α [HIF-1α], vascular endothelial growth factor, mitogen-activated protein kinase [MAPK], phosphorylated epidermal growth factor receptor, phosphorylated ERα [pERα]). 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.ResultsNinety-one patients (81%) attained a disease response, 48 achieved a complete clinical response (43%) whereas 22 did not respond (19%). Increased pERα 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-1α were significant factors for treatment resistance in all leave-one-out iterations. There was no significant interaction between these variables and treatment.ConclusionActivated ERα form was an independent factor for sensitivity to chemoendocrine treatment, whereas HIF-1α 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.

The OLTRE trial (ClinicalTrials.gov number: NCT02681562) is an open-label, ‘window of opportunity’ Phase II controlled trial to evaluate the biological activity of olaparib in locally advanced triple-negative breast cancer compared with other subtypes of locally advanced breast cancer patients carrying germinal BRCA mutation receiving olaparib with the same treatment approach. The primary end point is to investigate the correlation between baseline gene and protein expression profile in order to identify possible predictive markers of response to olaparib. The OLTRE trial is expected to identify the surrogate markers of the biological activity of olaparib in the treatment of patients with triple-negative breast cancer.

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-α 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-α-negative tumors in combination with immunotherapy approaches.

Persistent circadian rhythm of bone turnover in bone metastatic breast cancer suggests greater skeletal retention of bisphosphonates if administered in the night. We assessed differential effects of night vs morning administration of zoledronic acid (ZA) on bone turnover. Forty-four breast cancer patients with bone metastases were randomised to receive intravenous ZA (4 mg) at 1100 or 2300 hours every 28 days for four times. Urinary concentration N-telopeptide of type-I collagen (NTX) and deoxypyridinolines, and serum C-telopeptide of type-I collagen (CTX), bone alkaline phosphatase (ALP), osteocalcin and Parathyroid hormone (PTH) was measured in the morning at baseline and after 4, 7, 14, 28, 56 and 84 days. Urinary ZA concentration was also measured. Zoledronic acid caused significant decreases of NTX and CTX (P

The variability of serum osteoprotegerin (OPG) and soluble RANKL (sRANKL) along the 24-h cycle was assessed in 20 healthy women. No rhythmic variations of serum OPG, sRANKL or sRANKL/OPG ratio were detected as a group phenomenon. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL. INTRODUCTION: Physiological bone turnover shows diurnal variations. The aim of the study was to assess variability of OPG and sRANKL serum levels along the 24-h cycle. METHODS: Blood was collected from 20 healthy women (median age 31 years, range 25-65 years) at 4-h intervals between 08:00 and 24:00 and at 2-h intervals between 24:00 and 08:00. Serum albumin, cortisol, osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), OPG and total sRANKL were measured. Temporal variations were assessed by the COSINOR model. RESULTS: Circadian rhythms of cortisol and albumin documented a normal synchronization within the circadian structure. Serum OC and CTX showed rhythmic variations, peaking at night-time. Rhythmic variations of serum OPG, sRANKL and sRANKL/OPG ratio were not detected as a group phenomenon. On an individual basis, rhythmic changes were detected in ten patients for OPG and eight patients for sRANKL, with very small amplitudes and heterogeneous acrophases. CONCLUSIONS: The absence of consistent rhythmic variations of circulating OPG and sRANKL levels may reflect the absence of rhythmic variations of their expression in the bone microenvironment. Were this the case, the nocturnal rise of bone resorption should be accounted for by different, not RANKL/OPG-mediated factors. Since circulating OPG and sRANKL may derive from sources other than bone, rhythmicity could be masked by non-rhythmic or non-synchronized rhythmic expression in these sources. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL

// Manuela Milani 1, * , Sergio Venturini 2, * , Simone Bonardi 1 , Giovanni Allevi 1 , Carla Strina 1 , Maria Rosa Cappelletti 1 , Silvia Paola Corona 3 , Sergio Aguggini 1 , Alberto Bottini 1 , Alfredo Berruti 4 , Adrian Jubb 5 , Leticia Campo 5 , Adrian L. Harris 5 , Kevin Gatter 5 , Stephen B. Fox 6 , Daniele Generali 1, 7 and Giandomenico Roviello 7, 8 1 U.O. Multidisciplinare di Patologia Mammaria, U.S Terapia Molecolare e Farmacogenomica, ASST Cremona, Viale Concordia 1, Cremona, Italy 2 CE.R.G.A.S., Universita Bocconi, Milano, Italy 3 Peter MacCallum Cancer Centre, Bentleigh East VIC, Australia 4 U.O. Oncologia Medica, Spedali Civili si Brescia, University of Brescia, Brescia, Italy 5 Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK 6 Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia 7 Department of Medical, Surgery and Health Sciences, University of Trieste, Piazza Ospitale 1, Trieste, Italy 8 Department of Oncology, Medical Oncology Unit, San Donato Hospital, Italy * Manuela Milani and Sergio Venturini contributed equally to the study Correspondence to: Daniele Generali, email: daniele.generali@gmail.com Keywords: epirubicin resistance, haemoglobin, hypoxia-inducible factor, neoadjuvant, breast cancer Received: September 21, 2015 Accepted: July 18, 2017 Published: August 14, 2017 ABSTRACT 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.

INTRODUCTION: Recently the combination of the mammalian target of rapamycin (mTOR) inhibitor everolimus and the aromatase inhibitor exemestane has been shown to double the progression-free survival rate in advanced breast cancer. However, the effect of the interrelated pathways of hypoxia-inducible factor-1α (HIF-1α) and mTOR signaling, both of which are associated with a more aggressive breast cancer phenotype and endocrine resistance, on response in the neoadjuvant setting is unknown. We, therefore, have investigated the influence of these pathways with the aim of better defining those patients most likely to benefit from an endocrine-based therapy associated with/without mTOR inhibitors. PATIENTS AND METHODS: A total of 107 women with T2-4 N0-1 and estrogen receptor-positive breast cancer were randomly assigned to 6 months of primary letrozole (2.5 mg/daily) (LET) or LET plus oral "metronomic" cyclophosphamide (50mg/daily) (LET-CYC). Phospo-mTOR and HIF-1α were evaluated in tumor specimens collected before and after treatment using a tissue microarray format. RESULTS: LET-based therapy induced a downregulation of phospho-mTOR and HIF-1α expression (P = .0001 and P < .004, respectively). The reduction of HIF-1α expression observed was positively correlated with phospho-mTOR reduction (P < .03); however, no treatment interaction between the two proteins was detected. HIF-1α expression was significantly modulated by the treatment (P < .004) with a reduction both in the LET arm (45%, n = 36/80) (P = .05) and LET-CYC arm (55%, n = 44/80) (P = .04). HIF-1α reduction showed a relationship with clinical response confined in LET arm only (P < .03). CONCLUSIONS: In this neoadjuvant population, LET was able to modulate the phospho-mTOR and HIF-1α pathways and may define a subpopulation of nonresponders who may be most likely to benefit from mTOR inhibitors

Purpose: To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC). Methods: Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers. Results: Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by 18FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P

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.

Summary Purpose. To investigate whether magnetic resonance imaging (MRI) is superior to clinical palpation in the assessment of response of breast cancer to primary chemotherapy (PC). Patients and methods. Seventy-three patients with T2–4, N0, M0 breast cancer were treated with 3–4 cycles of single agent epirubicin before definitive surgery. MRI was performed at baseline condition and at the end of chemotherapy. Results. According to the WHO criteria, 20 (27.4%) patients attained a complete response (CR) by clinical palpation and 41 (56.2%) a partial response. The corresponding response rate by MRI was 11 (15.1%) and 34 (46.6%), respectively. Residual tumor assessed by MRI better correlated with pathologic measurements (Spearman r: 0.72) than residual tumor assessed by clinical palpation (Spearman r: 0.58). Post-chemotherapy histology evaluation revealed pathologic CR in three cases, only one of them was considered as complete responder by MRI. Residual disease consisted in in situ carcinoma in four cases, one of them was complete responder at MRI, the remaining three showed residual abnormal contrast enhancement indistinguishable from that of invasive tumors. Conclusions. As compared to pathology specimens, MRI is able to represent the extent of cancer more accurately than clinical palpation. It constitutes a promising technique in assessing the BC response to PC. The current limit of MRI is the scarce specificity in predicting the nature of residual disease.

Background: The objective of this study was to determine the optimal scheduling of 2.5 mg daily letrozole in neoadjuvant breast cancer patients to obtain pathological complete response (pathCR) and assess Ki-67 expression as an early predictor of response.Patients and methods:This single institution study comprised 120 oestrogen receptor (ER)-positive postmenopausal women with primary breast cancer (clinical stage ≥T2, N0-1), from three sequential cohorts (cohort A of 40, cohort B of 40 and cohort C of 40 patients, respectively) based on different duration of the neoadjuvant letrozole. Biological markers such as ER, progesterone receptor, HER2 and Ki-67 expression were tested at diagnosis and at definitive surgery.Results:A total of 89 patients (75.4%) achieved an objective response with 44 (37.3%) clinical CRs and 45 (38.1%) partial responses. The clinical CRs were significantly observed in cohort C (23 out of 40 patients, 57.5%) and B (16 out of 38 patients, 42.1%) compared with cohort A (5 out of 40 patients, 12.5%) (P-value for trend

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.

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.

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-1α 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 T2-4 N0-1 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-1α 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-1α. 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.

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

Purpose To investigate the activity of letrozole plus/minus oral metronomic cyclophophamide 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 antiangiogenetic effect and the combination of letrozole plus cyclophosphamide warrants testing in a randomized phase III trial.

Purpose: To investigate the relationship of hypoxia-inducible factor-1α (HIF-1α) 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-1α was assessed by immunohistochemistry in 187 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 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-1α (P < 0.05), and HIF-1α was an independent predictor of response (P < 0.048). HIF-1α 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-1α-negative tumors. Conclusions: HIF-1α 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-1α expression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies.

This study was designed to address whether simultaneous primary chemo-hormonal therapy provides additional activity compared with chemotherapy alone in breast cancer patients with operable or locally advanced disease. Between January 1997 and January 2002, 211 consecutive patients with T2–4, N0–1, M0 breast cancer were randomized to receive either epirubicin alone (EPI) or epirubicin plus tamoxifen (EPI-TAM). Ki67 expression was evaluated immunohistochemically in tumor specimens obtained before chemotherapy by incision biopsy and at definitive surgery. Tumor shrinkage of >50% was obtained in 76% of patients randomized in the EPI arm and 81.9% of patients randomized in the EPI-TAM arm (not significant). The corresponding rates of clinical and pathological complete response were 20.2 and 21.9% (not significant), and 4.8 and 6.7% (not significant), respectively. Pathologically complete response was more frequently observed in estrogen receptor (ER)-negative (ER -) tumors (P=0.04) and correlated with elevated baseline Ki67 expression (P

The response to primary chemotherapy is an important prognostic factor in patients with non metastatic breast cancer. In this study we compared the assessment of response performed by clinical palpation to that performed by echography and mammography in 141 out of 157 consecutive breast cancer patients (T2-4, N0-1, M0) submitted to primary chemotherapy. A low relationship was recorded between tumor size assessed clinically and that evaluated by either mammography: Spearman R = 0.38 or echography: R = 0.24, while a greater correlation was found between the tumor dimension obtained by the two imaging techniques (R = 0.62). According to the WHO criteria, the grade of response of breast cancer to primary chemotherapy, showed by mammography and echography, was less marked than the grade of response seen at clinical examination. Residual tumor size assessed clinically depicted a stronger correlation with pathological findings (R = 0.68) than the residual disease assessed by echography (R = 0.29) and mammography (R = 0.33). Post-chemotherapy histology evaluation revealed pathological complete response in three cases (2.1%). Two of these cases were judged as complete responders by clinical palpation but only one was recognized by mammography, and none by echography. Clinical response, but not the response obtained by the two imaging techniques, was a significant predictor for longer disease free survival (p = 0.04). To conclude, physical examination measurements remain the method of choice in evaluating preoperatively the disease response in trials of primary chemotherapy. Prediction of pathological outcome is not improved by echography and mammography.

e11546 Background: nPLD has the same clinical benefits but less toxicity than the traditional doxorubicin. It’s well reported mC produces anti-proliferative and anti-angiogenic effect in tumours. We conducted a retrospective study to evaluate the safety and activity of the association of nPLD and mC in pts with locally advanced BC. Methods: A total of 41 female pts with HER2-negative locally advanced BC unfit for standard chemotherapy received nPLD 25 mg/mq biweekly for eight courses along with mC 50 mg daily as primary systemic treatment. Toxicity and clinical-radiological response with Ki67 and CD31 expression were evaluated in tumour specimens obtained before and after treatment. Results: Pts characteristics included: median age 47.5 years; 31.7% Luminal A (LA), 56.1% Luminal B (LB), and 12.2% Triple Negative BC subtype classified by immunohistochemistry. The rate of breast-conserving surgery was 59.5%. All pts were evaluable for response. Objective response rate (ORR) was 58.5% (95% confidence interval, 42.1% to 73.7%), with 4 (9.7%) clinical complete responses (CR) and 20 (48.8%) partial responses. Sixteen pts (39.0%) achieved a stable disease and 1 pt progressed. One pt experienced a pathological CR. LA subtype was significantly associated with better ORR than others subtypes (p=0.049) and especially than LB (p=0.040). There was a borderline significant reduction of Ki67 expression after treatment (median, 15.5% to 9%; p=0.056), but no significant variation in CD31 expression (p=0.865). The majority of adverse events (AEs) were mild or moderate. Grade ≥3 AEs included neutropenia (12.2%), vomiting (4.9%) and hyperglycemia (2.44%). Temporary treatment suspension due to toxicity occurred in 8 (19.5%) pts; discontinuation was necessary in 1 pt. No symptoms were related to impairment of cardiac function. Mean left ventricular ejection fraction levels did not change substantially before and after treatment: 58.7% and 57.1%, respectively. Conclusions: The combination of nPLD and mC is well tolerated, with a promising activity as preoperative therapy in unfit pts with locally advanced BC.

8108 Background: Letrozole has repeatedly found to be active as primary endocrine therapy in breast cancer patients. The aim of the study was to explore the activity of single agent Letrozole or Le...

766 Background: Response to chemotherapy in metastatic breast cancer patients is recognised as an important prognostic factor and may lead to a better selection of patients to be addressed to surgery. Methods: From 1990 to 2001, 328 consecutive patients with metastatic breast cancer were submitted to systemic therapy with anthracycline containing regimens. One hundred fifty-seven patients (48.3%) had bone metastases, 130 (40.0%) lung, 93 (28.0%) liver, 123 (37.7%) skin/lymphnodes, 3 (12.5%) brain and 16 (4.9%) other sites of disease. One hundred ninety-two (58.5%) patients had one site of disease, and 136 (41.5%) two or more. Results: Fifty-six patients (17.4%) attained a complete response (CR), 141 (43.9%) a partial response (PR), 72 (22.4%) a stable disease (SD), while 31 (9.7%) patients progressed (PD). All patients were evaluated for surgery of residual disease and nineteen (5.8%) were radically resected (1 showing a CR, 15 a PR, 2 a SD and 1 a PD disease). Nine (47.4%) of them had liver metastases, 6...

Aim: Treatment of triple-negative breast cancer (TNBC) imposes great challenges, due to a lack of molecular targets. While use of gonadotropin-releasing hormone (GnRH) analogs has been validated in ER-positive breast cancer, this option has not been investigated in TNBC, even though a significant portion of these tumors upregulate GnRH receptors. We performed a meta-analysis of the literature to evaluate the effect of GnRH analogs in TNBC. Methods: Four studies were included in this study. Results: We detected a non-significant improvement in overall survival with GnRH analogs, while progression-free survival was unchanged. Discussion: The majority of the trials evaluated in this analysis were designed to test efficacy of GnRH analogs in preventing premature ovarian failure. This may represent a limitation of our study as these trials were not specifically designed to detect differences in survival outcome measures. Conclusion: Our results suggest that GnRH analogs may be useful as a targeted therapy in TNBC. Randomized prospective clinical trials are needed to investigate this hypothesis in the clinic.