Pre-surgical effects of serms, anti-COX-2 and aromatase inhibitors in breast cancer patients

INTERVENTION: Breast cancer patients are randomized based on menopausal status to Raloxifene or Tamoxifen or placebo, to Exemestane or Celecoxib or placebo. Subjects with histologically‐confirmed ER positive primary breast cancer will be randomly assigned in a 2:2:1 ratio to receive treatment or matching placebo for a total period of 6 weeks prior to surgery. Specifically, 125 premenopausal women will be randomized to either low‐dose tamoxifen (10 mg/week) or raloxifene 60 mg/day or placebo, while 125 postmenopausal women will be allocated to exemestane (25 mg/day) or celecoxib (800 mg/day) or placebo. Raloxifene: Evista® 60 mg/tablet. Daily administration. Tamoxifen: Nomafen® 10 mg/tablet. Weekly administration. Exemestane (Aromasin®): 25 mg/tablet. Daily administration. Celecoxib: (Celebrex®) 200 mg/tablet. Two tablets will be administered twice a day Placebo formulation of size‐matched tablets will be manufactured by an external pharmaceutical service. At randomization each subject will receive 1 box with 8 weekly bottles of the drug and will be instructed to take 1 tablet per day. However, in order to maintain the blinding of a weekly dose of tamoxifen in the premenopausal group, each subject will receive also 8 blisters with 1 tablet each. She will be asked to take 2 tablets the first day of the week, 1 from the bottle and 1 from the blister, and then proceed with 1 tablet/day for the rest of the week. Blisters will contain placebo or tamoxifen accordingly. A regards the post menopausal group, given the unnecessary use of 3 extra placebo tablets, double‐blinding will be maintained for exemestane and placebo (1 tablet/day) only, while the celecoxib arm will be open (2 tablets twice a day). CONDITION: Estrogen receptor positive breast cancer ; Cancer ; Malignant neoplasm of breast PRIMARY OUTCOME: The primary endpoint is the change (tru‐cut biopsy at baseline, and surgical specimen at surgery) in the percentage of neoplastic cells expressing the proliferation antigen Ki‐67.; ; Biopsy and surgical specimens are fixed in 10% neutral‐buffered formalin for 6‐8 hours before being embedded in paraffin. Sections (4‐micron thick) are cut and stained with hematoxylin and eosin. Consecutive serial sections are used for immunohistochemical determinations. Expression Ki‐67 will be determined by immunohistochemistry. Briefly, de‐waxed tumor sections are pretreated with 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase activity and then treated with a solution of 0.001 M EDTA (pH 8.0) at 99°C for 20 minutes to retrieve antigens. The tumor sections are then incubated with primary mouse monoclonal antibodies Ki‐67 (clone Mib‐1, 1:200 dilution).; ; Primary endpoints will be detected at baseline and after 6 weeks of treatment (at biopsy and at surgery) INCLUSION CRITERIA: 1. Female, aged at least 18 years old, no upper age limit 2. Performance status = 0 (SWOG) 3. Histologically‐confirmed ER+ primary breast cancer candidated for conservative surgery 4. Stage T1‐2, N0‐1, M0 or women with larger tumors who refuse chemo and/or endocrine therapy before surgery 5. No previous treatment for breast cancer 6. Provision of written informed consent SECONDARY OUTCOME: Secondary endpoints: Change in ER and PgR expression and apoptosis as measured by Caspase‐3. Change in markers of hemostasis and inflammation: fibrinogen, antithrombin III (AT III), plasminogen activator inhibitor‐I (PAI‐I), ultrasensitive C‐reactive protein (US‐CRP). Change in circulating hormones (estradiol, estrone, estrone‐sulphate, and sex hormone binding globulin). DNA will be extracted from circulating lymphocytes and some genetic polymorphisms related to hormone and drug metabolism will be investigated.; ; Expression of ER, PgR, Her2/neu, and Caspase‐3 will be determined by immunohistochemistry. The tumor sections are then incubated with primary mouse monoclonal antibodies to ER (clone 1D5, 1:100 dilution), PgR (clone 1A6, 1:800 dilution), or with rabbit polyclonal antibody to the Her2/neu protein (1:3200 dilution), and to Caspase‐3 (clone CPP32).; Routine hematology and biochemistry assessments will be performed at the central laboratory of the institute.; IGF‐I levels will be determined from plasma by a chemiluminescent immunometric assay; antithrombin‐III will determined by a kinetic colorimetric method. Serum concentration of US‐CRP, SHBG and IGFBP‐3 will be measured by a two‐site chemiluminescent enzyme immunometric assay. Serum C‐telopeptide and osteocalcin will be determined with a electrochemiluminescent immunometric assay. Serum estradiol and estrone‐sulphate will be determined by the use of commercially available ultrasensitive radioimmunoassay kits.; Genomic DNA will be extracted from frozen whole blood samples stabilized with EDTA by the use of a commercially available kit (QIAamp DNA Blood kit) purchased from Qiagen S.p.A, Milan, Italy).The evaluation of the CYP2D6 single nucleotide polymorphisms will be performed utilizing a semiautomated instrument (Light Cycler ? Roche) that allows more rapid assay procedures and guarantees a good product specificity. Specific primers and probes are labeled with fluorescent dyes and light emission monitored to detect genotyping.; ; Secondary endpoints will be detected at baseline and after 6 weeks of treatment (at biopsy and at surgery)