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BACKGROUND: Matrix metalloproteinases (MMP) play an essential role in tissue remodelling and menstruation and appear to be regulated by cytokines such as interleukin-1a (IL-1a). In order to investigate their role in the pathogenesis of endometriosis, the aim of the present study was to compare the protein localization of matrix metalloproteinase-1 (MMP-1) and of its main stimulatory cytokine IL-1a in eutopic and dystopic endometrium of patients with endometriosis. METHODS: MMP-1 and IL-1a protein localization was analysed retrospectively in paired paraffin-embedded tissue biopsies obtained simultaneously from the endometrial cavity and from endometrial lesions of 37 patients with peritoneal or ovarian endometriosis and in cycling endometria from 37 women without endmetriosis. Protein localization was demonstrated by immunohistochemistry; antibody specifity was confirmed by western blot analysis. RESULTS: MMP-1 and IL-1a protein staining in women suffering from endometriosis was significantly more pronounced in endometriotic lesions than in eutopic endometrium. This held true for both epithelial MMP-1 and IL-1a staining (P<0.006 and P<0.001), and for stromal MMP-1 and IL-1a staining (P<0.001 and P<0.001). Furthermore, stromal MMP-1 and IL-1a were significantly co-expressed in dystopic endometriotic tissue (P=0.045). Endometrial MMP-1 and IL-1a protein expression pattern in eutopic endometrium from women suffering from endometriosis, however, did not differ significantly from the pattern seen in healthy women. CONCLUSIONS: The increased expression of both matrix-degrading MMP-1 and its major stimulatory cytokine IL-1a in endometriotic lesions and the selective co-expression in the stroma of endometriotic foci clearly suggests their involvement in the pathogenic mechanisms leading to local invasion and tissue destruction. [ABSTRACT FROM AUTHOR]

Over-expression of members of the ErbB-receptor family has been associated with malignant transformation. The amplification of Her-2/neu in tumor tissue is now an established prognostic factor in breast cancer. In order to initiate signal transduction, ErbB-receptor monomers need to form homo- or heterodimers. The composition of these dimers is thought to influence both quality and quantity of downstream signaling pathways, and to determine the biological response. We have investigated the protein expression pattern of the four ErbB-receptors EGFR, Her-2/neu, Her-3 and Her-4, and correlated it with their putative ligands EGF, TGF-α and HRG in 74 women with invasive breast cancer. Using western blot-analysis on cell membrane isolates, we detected the co-expression of all four ErbB-family members in 79.7% of cases, and of all of the three investigated ligands in 82.4%. We did not observe a correlation between EGFR and Her-2/neu or Her-4 protein expression, EGFR and Her-3 (p = 0.005), and Her-3 and Her-4 (p = 0.05) were clearly co-expressed. The strongest overall correlation, was found between Her-2/neu and Her-3 (p < 0.001) and between Her-2/neu and Her-4 (p = 0.001). This was particularly true in nodal-positive tumors (p <0.001 and p = 0.002) whereas in nodal-negative tumors the co-expression was either less significant (Her-2/neu and Her-3; p = 0.01) or not significant (Her-2/neu and Her-4). The co-expression of EGFR/Her-3 was associated with the expression of all ligands, whereas the Her-2/neu/Her-3 was correlated with HRG (p = 0.002), thereby indicating a functional relation between specific receptor-dimer combinations and putative ligands. Taken together, we have performed the first comprehensive survey of ErbB-system expression in breast cancer, and have demonstrated the presence of a co-regulated receptor/ligand system in vivo. We have further shown that Her-2/neu is the preferred co-expression partner in nodal-positive tumors and thus the most likely dimerization candidate in malignant breast tumors. [ABSTRACT FROM AUTHOR]

Estetrol (E4) is a fetal estrogen with estrogenic effects on reproductive organs and bone in preclinical models and in postmenopausal women. However, E4 exerts antiestrogenic effects on breast cancer (BC) cell growth in vitro and in vivo. We have investigated the effect of 14 days preoperative treatment with 20mg E4 per day on tumor proliferation markers, sex steroid receptor expression and endocrine parameters in a prospective, randomized, placebo-controlled, preoperative window trial in 30 pre- and post-menopausal women with estrogen-receptor positive early BC. E4 had a significant pro-apoptotic effect on tumor tissue, whereas Ki67 expression remained unchanged in both pre- and post-menopausal women. E4 increased sex-hormone-binding globulin significantly thereby reducing the concentrations of bioavailable estradiol. Follicle-stimulating hormone levels decreased in postmenopausal women only and luteinizing hormone levels remained unchanged. Systemic insulin growth factor-1 levels decreased significantly. Intratumoral epithelial ERα expression decreased significantly and a trend was found towards an increased expression of ERβ. This clinical data support the preclinical findings that E4 has antiestrogenic effects on BC cells, whereas earlier studies have shown that E4 has estrogenic effects on reproductive tissues and bone. Further clinical studies seem acceptable and are needed to confirm the safety and efficacy of E4 for the breast in hormone replacement therapy, including hormone replacement therapy in women who have or have had BC, especially in those BC patients treated with aromatase inhibitors and suffering from serious complaints due to estrogen deficiency., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Despite patient selection based on ERBB2 overexpression, not all patients benefit from trastuzumab therapy. We have investigated whether a ERBB2 gene dosage effect might provoke increased biological aggressiveness and altered trastuzumab sensitivity. Absolute ERBB2 copy numbers ("CN") and ERBB2/centromer 17 ratios ("R") were measured by FISH analysis in tumors of 127 patients receiving trastuzumab-based treatment for Her-2/neu overexpressing metastatic breast cancer. CN and R were both significantly associated with shorter time to first metastasis (TTM) (CN: OR: 1.099, 95% CI: 1.042-1.159; R: OR: 1.211, 95% CI: 1.080-1.357) and longer PFS (CN: OR: 0.917, 95% CI: 0.867-0.969; R: OR: 0.840, 95% CI: 0.743-0.949) in a continuous variable Cox's regression model. Tumors with ERBB2/centromer 17 ratios of <2.2 had a significantly shorter TTM (p = 0.002) and significantly longer PFS (p = 0.003) than tumors with low-level (R: 2.2-6) and high-level amplification (R: >6). Interestingly, when ERBB2 copy numbers were analyzed, a significantly shorter TTM (p = 0.001) and longer PFS (p = 0.026) were observed in the group with high-level amplified CN (CN: >13), while no difference was observed between non- and low-level amplified CN. R, but not CN, was an independent predictor of complete (CR; OR: 1.685; 95% CI: 1.122-2.532) and partial (PR; OR: 1.704; 95% CI: 1.136-2.556) response in logistic regression analysis. CR (p = 0.016) rates were significantly higher in the high-level amplification group (R > 6), but no difference existed in response rates between non- and low-level amplified tumors in Chi-square tests. High-level ERBB2 amplification is associated with shorter TTM, but improved response to trastuzumab in metastatic breast cancer., (© 2013 UICC.)

BRCA mutation carriers are at high risk of developing ovarian cancer. Ovarian malignancies are usually identified at an advanced stage with poor prognosis, attributed to inadequate options of early detection. Because of its risk-reducing effect of nearly 96%, prophylactic salpingo-oophorectomy is still the leading option for risk-reduction in women with a positive BRCA mutation status. The presence of ovarian cancer precursor lesions, such as epithelial inclusion cysts (EICs) or cortical invaginations (CIs), has previously been discussed in several studies with diverse conclusions. We retrospectively investigated a large and consistent population (n=94) of BRCA mutation carriers for the presence of potential preneoplastic and neoplastic changes. We also examined the role of specific lifestyle factors. Ninety-four women with disease-associated germline BRCA1 or BRCA2 mutations were included in this retrospective study. All women had undergone genetic counseling and prophylactic salpingo-oophorectomy, which was performed at a mean age of 43.33 years (range 27-66). Histological slides of both ovaries were reviewed by an independent pathologist. Data concerning lifestyle factors were collected from medical files and questionnaires. Two malignant lesions (2.1%), one bilateral serous papillary adenocarcinoma of the epithelial surface and one adenocarcinoma of the peritoneum with involvement of the left adnexa, and one lesion (1.1%) with obvious malignant potential, one mucinous borderline tumor of the right ovary, have been identified. We registered a high prevalence of CIs (30; 31.9%) and EICs (44; 46.8%) in prophylactically removed ovaries of BRCA mutation carriers. A significant correlation (P=0.002) was found with regard to the presence of EICs in women with increased BMI. Concerning the regular consumption of alcohol as a risk factor for premalignant lesions, in particular CIs, a statistically insignificant trend (P=0.083) was noted. Overweight women seem to be at risk of developing more cortical invaginations than women of normal weight. To improve the final outcome of the disease, women at increased risk of ovarian cancer should be appropriately informed of potential increased risk factors.

The eLEcTRA trial compared efficacy and safety of letrozole combined with trastuzumab to letrozole alone in patients with HER2 and hormone receptor (HR) positive metastatic breast cancer (MBC). Patients were randomized to either letrozole alone (arm A, n = 31) or letrozole plus trastuzumab (arm B, n = 26) as first-line treatment. Additional 35 patients with HER2 negative and HR positive tumors received letrozole alone (arm C). Median time to progression in arm A was 3.3 months compared to 14.1 months in arm B (hazard ratio 0.67; p = 0.23) and 15.2 months in arm C (hazard ratio 0.71; p = 0.03). Clinical benefit rate was 39% for arm A compared to 65% in arm B (odds ratio 2.99, 95% CI 1.01-8.84) and 77% in arm C (odds ratio 5.34, 95% CI 1.83-15.58). The eLEcTRA trial showed that the combination of letrozole and trastuzumab is a safe and effective treatment option for patients with HER2 positive and HR positive MBC., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: The Livial Intervention Following Breast Cancer: Efficacy, Recurrence and Tolerability Endpoints (LIBERATE: Clinical http://Trials.gov number NCT00408863), a randomized, placebo-controlled, double-blind trial that demonstrated that tibolone (Livial), a tissue-selective hormone-replacement therapy (HRT), increased breast cancer (BC) recurrence HR 1.40 (95% CI, 1.14 to 1.70; P = 0.001). A subgroup of women was entered into a study of bone mineral density (BMD)., Methods: Women with surgically excised primary BC (T1-3, N0-2, M-0) within the last 5 years, complaining of vasomotor symptoms, were assigned to tibolone, 2.5 mg daily, or placebo treatment for a maximum of 5 years. The BMD substudy enrolled 763 patients, using dual-energy X-ray absorptiometry (DXA) scanning at baseline and at 2 years., Results: In the bone substudy, 699 of 763 women were eligible (345 allocated to tibolone, and 354, to placebo). After undergoing DXA scans, 300 (43%) women had normal BMD; 317 (45%), osteopenia; and 82 (11.7%), osteoporosis. Low body-mass index (P < 0.001), Asian race (P < 0.001), and late age at menarche (P < 0.04) predicted low bone mass at baseline. Tibolone increased BMD by 3.2% at the lumbar spine and 2.9% at the hip compared with placebo (both P < 0.001). The majority of fractures (55%) occurred in osteopenic patients. Women with normal BMD had increased recurrence with tibolone, 22 (15.6%) of 141 compared with placebo, 11 (6.9%) of 159 (P = 0.016), whereas no increased BC recurrence was seen in women with low BMD; 15 (7.4%) of 204 taking tibolone versus 13 (6.7%) of 195 taking placebo., Conclusions: Tibolone is contraindicated after BC treatment, as it increases BMD and BC recurrence. Risk of BC recurrence was elevated in BC women with normal BMD (compared with low) who took tibolone.

Background: BRCA-1/2 germline mutations are responsible for early onset breast cancer and familial association. The underlying causes of the characteristic phenotypic behavior are not completely understood, but mammary stem cells appear to have a key role in this process., Materials and Methods: We have investigated the presence of mammary stem / progenitor cells in normal tissues and in tumor tissues obtained from women with and without BRCA1/2 germline mutations by utilizing ALDH-1 immunohistochemistry., Results: Isolated ALDH-1 positive cells were found in 15/28 (54%) of breast cancer samples from women with BRCA 1 or 2 mutations and in 33 /51 (65%) of matched sporadic breast cancer cases (p=0.5949, Chi Square test). While mammary stem cells were also detected in non-malignant breast lesions, only 41% of the tissues contained ALDH-1 positive cells (p=0.0371, Chi Square test). In patients with BRCA germline mutations ALDH-1 positive cells were more common in p53 positive (p=0.0028, Chi Square test) tumors, in high grade (p=0.0796), and in larger tumors (p=0.0604), while no such association was seen in sporadic cancer cases. In our patients, the expression of ALDH-1 positive cells in breast cancer was neither associated with disease-free and overall survival, nor time to metastasis., Conclusion: Breast cancers from BRCA mutation carriers do not harbor more ALHD-1 positive cells than sporadic tumors, and their more aggressive phenotype can thus not be explained by an increased stem cell pool. The presence of ALDH-1 in normal breast tissue suggests that additional factors determine the biological behavior of mammary stem cells.

Background: Climacteric symptoms such as hot flushes and vaginal dryness are very common in breast cancer patients, resulting either from age or adjuvant therapy. Tibolone, a synthetic steroid, is effective in reducing these symptoms in healthy post-menopausal women, but this has never been studied in a large breast cancer population., Objectives: The primary objective of LIBERATE trial was to study safety of tibolone 2.5mg daily versus placebo as primary, in symptomatic breast cancer survivors. The aim of this present paper was to report effects of tibolone on climacteric symptoms, vaginal dryness and health-related quality of life in the study population. This trial is registered with ClinicalTrials.gov, n. NCT00408863., Methods: The trial was conducted between June 2002 and July 2007. Concerning quality of life variables, a daily Diary Cards during the first three months and the Climacteric Symptoms Form and at each visit were used to register frequency and intensity of hot flushes. Mean vaginal dryness scores were calculated on the basis of individual ratings at baseline and at week 104. A subset of patients assessed their quality of life filling in the Women's Health Questionnaire (WHQ)., Results: Of the 3148 women recruited, 3133 received trial medication (1575 in the tibolone group and 1558 in the placebo group). The median duration of treatment was 2.75 years. In total 3098 women (1556 on tibolone, 1542 on placebo) were included in the intention-to-treat (ITT) population for efficacy analysis. Data on vaginal dryness are available for 2144 patients and 883 women (438 on tibolone, 445 on placebo) answered to WHQ. The mean change in number of hot flushes per day was 2.74 (43.1%) in the tibolone group and -1.77 (-27.5%) in the placebo group (p<0.0001) at week 12 and -4.62 (-65.6%) on tibolone as compared to -3.73 (-52.5%) on placebo (p<0.0001) at week 104. For the composite score the mean changes at week 12 were -0.19 (-10.6%) and -0.14 (-7.7%), respectively (p=0.0006). Vaginal dryness score improved at week 104 in the tibolone group as compared to placebo (-0.46 versus -0.29, respectively; p<0.0001). Across the assessments up to two years with WHQ, tibolone was more effective than placebo in improving sexual health, sleep quality and mood domains. Women using tamoxifen showed less improvement in climacteric symptoms with tibolone, than women only receiving tibolone without any adjuvant therapy., Conclusion: The results of the LIBERATE trial show that tibolone is effective in symptomatic breast cancer patients and improves their quality of life. However, this finding should be judged within the context of the main outcome of the trial, showing that tibolone increases the risk of recurrence. The use of tibolone in women with breast cancer will remain contraindicated and any off-label use incurs a now proven risk., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Sentinel node biopsy is a widely accepted alternative to primary axillary lymph node dissection for ipsilateral nodal assessment in breast cancer. We have performed a retrospective chart review in 713 consecutive patients with primary, operable breast cancer who underwent sentinel node biopsy in order to identify factors that determine the sentinel node identification rate. Chi-squared test, univariate and multivariate analyses were used to evaluate the influence of different factors on the sentinel identification rate. Among the factors investigated, tumour size was correlated with sentinel lymph nodes detection rates (multiple logistic regression, P= 0.002). In addition, the patient's age showed to be a significant influencing factor (multiple logistic regression, P= 0.006). Body mass index and grade only exhibited a significant correlation with the identification rate in the univariate (P= 0.041, P= 0.025), but not in the multivariate analysis (P= not significant). All associations were found to be independent of the site of injection. Interestingly, surgeons with intermediate expertise (11-20 prior dissections) had the highest detection rates (P= 0.004). We conclude that sentinel identification rates are higher in larger tumours and in younger patients, independent of the injection site. Surgical experience in sentinel node dissection is not linearly correlated with higher identification rates., (© 2011 Blackwell Publishing Ltd.)

Patients with hormone receptor positive breast cancer who are treated with endocrine therapy generally have a good prognosis. However, resistance to hormonal therapy and progression occurs, and the reasons for this are manifold. It has been proposed that the local estrogenic environment has a role in the process of local invasion and progression. We have determined the expression pattern of estrogen receptor α, estrogen receptor β, and the epithelial and stromal expression of the estrogen-metabolizing enzymes aromatase and sulfotransferase by immunohistochemistry in tissue arrays, containing 50 paraffin-embedded sets of tissues obtained from breast cancer and from corresponding metastatic axillary lymph nodes of the same patients. We have found statistically significant higher estrogen receptors α and β expression in primary tumors than in corresponding lymph node metastases (p = 0.0004 and p = 0.003, respectively). Aromatase was also expressed more frequently in epithelial as well as in stromal cells of the malignant tumor when compared to according lymph node metastases (p = 0.08 and p = 0.12, respectively). While in lymph node metastases only estrogen receptor α and stromal aromatase expression were correlated (p = 0.01), significant associations were seen between the estrogen receptor β and sromal aromatase, and epithelial sulfotransferase (p = 0.0006 and p = 0.03, respectively) in the primary tumor. We hypothesize that the decreased expression of local estrogens by aromatase, in combination with a decreased expression of estrogen receptors α and β in lymphatic metastases, renders these metastases hormone insensitive and could contribute to the poor response to endocrine therapy that is often seen in nodal-positive tumors.

Objective: Aromatase inhibitors are essential as endocrine treatment for hormone receptor-positive postmenopausal breast cancer patients. Menopausal symptoms are often aggravated during endocrine treatment. We investigated whether vaginal estriol is a safe therapeutic option to overcome the urogenital side-effects of aromatase inhibitors. Serum hormone levels were used as the surrogate parameter for safety., Methods: Fasting serum hormone levels of ten postmenopausal breast cancer patients receiving aromatase inhibitors were prospectively measured by electro-chemiluminescence immunoassays and gas chromatography/mass spectrometry before and 2 weeks after daily application of 0.5 mg vaginal estriol (Ovestin® ovula), respectively., Results: Two weeks of daily vaginal estriol treatment did not change serum estradiol or estriol levels. However, significant decreases in levels of serum follicle stimulating hormone (p = 0.01) and luteinizing hormone (p = 0.02) were observed. Five out of six breast cancer patients noticed an improvement in vaginal dryness and/or dyspareunia., Conclusions: The significant decline in gonadotropin levels, indicating systemic effects, has to be kept in mind when offering vaginal estriol to breast cancer patients receiving an aromatase inhibitor.

Preliminary studies have shown that systemic beta-human chorionic gonadotrophin (betaHCG) therapy alleviates endometriosis-related chronic pelvic pain. The underlying mechanism, however, is completely unknown. This study has investigated the dose-dependent alterations in the overall gene expression profile of endometriosis-derived stromal cells under increasing concentrations of betaHCG by using the Affymetrix GeneChip U133 Set. It has been previously shown that betaHCG concentrations of 0.1U/ml and higher lead to a significant and dose-dependent increase in the expression of 68 genes. This study reports on a cluster analysis which identified three clusters of genes with a comparable expression pattern in response to increasing concentrations of betaHCG. Most of the up-regulated genes encoded proteins that are involved in cell adhesion, intercellular communication, extracellular matrix remodelling, apoptosis and inflammation. Stromal monocultures from eight patients, treated with and without 50U/ml of betaHCG, were then incubated and real-time polymerase chain reaction for the highly up-regulated genes PAI2, DUSP6, PLAU and MMP1 performed in order to validate the cDNA array findings in patients with endometriosis. Taken together, this study shows that betaHCG induces dose-dependent characteristic response clusters in the gene expression profile of stromal cells obtained from endometriotic lesions which could explain the differential biological responses of betaHCG in endometriosis., (Copyright (c) 2010. Published by Elsevier Ltd.)

Objective: Adiponectin serum levels have been shown to be inversely correlated with breast cancer risk. The protein is believed to act through adiponectin receptor 1 (AdipoR1) and has been suggested to play an important role in cancer development. While AdipoR1 is known to be expressed in invasive tumors, its role in DCIS remains elusive. We therefore investigated AdipoR1 expression in both invasive and preinvasive breast cancer., Methods: Tissue microarrays were established from paraffin-embedded archived tissues which contained 104 invasive breast cancers with adjacent preinvasive component (DCIS) as well as 96 preinvasive breast cancers. AdipoR1 expression was investigated by immunohistochemistry and correlated with clinical and tumor parameters., Results: AdipoR1 was detected in stromal and epithelial components of both invasive and preinvasive breast cancer. However, stromal and epithelial immunoreactivity for AdipoR1 was significantly higher in invasive breast cancer compared to preinvasive DCIS (p<0.001 and p=0.009). Within DCIS, AdipoR1 expression was inversely correlated with tumor size (r=-0.238, p=0.033). Menopausal status showed no influence on AdipoR1 expression., Conclusions: The altered expression of AdipoR1 in invasive breast cancer compared to DCIS suggests that the receptor-binding protein adiponectin might exert growth inhibitory effects that are overcome in transformation of preinvasive to invasive breast cancer., (Copyright 2009 Elsevier Inc. All rights reserved.)

Purpose: Previously, we have shown that p27 may be a potential predictive biomarker for the selection of premenopausal women with early-stage hormone-responsive breast cancer for adjuvant endocrine therapy. The purpose of the present study was to assess the clinical relevance of p27 expression in postmenopausal hormone receptor-positive breast cancer patients who were treated with adjuvant tamoxifen therapy., Experimental Design: We determined the expression of p27 by immunohistochemistry in the surgical specimens of breast carcinoma patients who had been enrolled in Austrian Breast and Colorectal Cancer Study Group Trial 06 and received tamoxifen for 5 years. Early relapse and death within the first 5 years of follow-up were analyzed using Cox models adjusted for clinical and pathologic factors., Results: p27 expression was high (>70% p27-positive tumor cells) in 252 of 483 (52%) tumor specimens and was associated with favorable outcome of the patients. Women with high p27 expression had a significantly longer disease-free survival (adjusted hazard ratio for relapse, 0.22; 95% confidence interval, 0.11-0.42; P < 0.001) and overall survival (adjusted hazard ratio for death, 0.39; 95% confidence interval, 0.21-0.72; P = 0.002) as compared with women with low p27 expression., Conclusion: Low p27 expression independently predicts early relapse and death in postmenopausal women with early-stage, hormone receptor-positive breast cancer who received adjuvant tamoxifen for 5 years.

Objectives: Tamoxifen is a partial ER antagonist that is highly effective in the treatment of receptor positive breast cancer. It significantly reduces recurrence and improves survival in both pre- and postmenopausal women. Unfortunately, many ER+ positive tumors progress despite tamoxifen treatment and until now, no possibility exists to prospectively identify tamoxifen-resistant tumors. It has been suggested that that in HER2 over-expressing tumors, cross-talk via activated HER2 receptors is a key mechanisms by which tumors become tamoxifen-resistant., Methods: We have therefore used immunohistochemistry to analyze the expression of HER2 and activated ptyr-1248 HER2 in 408 women of ER+, early breast cancer who had received at least 2 years of adjuvant tamoxifen. We then analyzed possible associations between HER2 and pHER2 expression, and prognostic parameters, and evaluated the effect of HER2 expression and survival., Results: With HER2 being positive in 12 of 208 (2.9%) of ER+ positive tumors, HER2 overexpression was found to be considerably less common in ER+ tumors than what has been thought previously. The majority of HER2 overexpressing tumors, however, also expressed the activated receptor form (r = 0.664; P < 0.0001). Both HER2 and pHER2 are moderately correlated with Grading (r = 0.138; P = 0.0052 and r = 0.118; P = 0.0241, respectively) and nodal involvement (r = 0.163; P = 0.0018 and r = 0.134; P = 0.016, respectively), but neither HER2 nor its activated form are significant predictors of RFS, DFS, or OS., Conclusions: Taken together, we have demonstrated that in ER+ breast cancer, the HER2 receptor is commonly activated, but its low prevalence in ER+ tumors does not render it a useful prognostic parameter in tamoxifen-treated patients.

Purpose: Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001)., Patients and Methods: Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry., Results: Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo., Conclusion: Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.

ERBB2 amplification and consecutive overexpression is a predictor for poor prognosis in breast cancer patients. In addition, incomplete resection of ERBB2-overexpressing tumors leads to increased proliferation of residual breast cancer cells. While the local release of cytokines is thought to be responsible for the malignant behavior of remaining tumor tissue, the exact mechanism is still unknown. We have analyzed epidermal growth factor receptor (EGFR), activated (p)EGFR, and activated (p)ERBB2 protein expression in ERBB2-overexpressing and in non-ERBB2-overexpressing tumors from patients who underwent breast surgery and consecutive re-excision for involved margins, and compared expression levels by immunohistochemistry. While overall ERBB2 protein expression in the initial and the re-excised sample were comparable, we observed an increase in pERBB2 in ductal carcinomas in situ in both, ERBB2-overexpressing (16/21 vs 24/24; P=0.018, chi(2) test) and non-ERBB2-overexpressing tumors (3/28 vs 5/12; P=0.025, chi(2) test). pERBB2 was not increased in invasive tumors, regardless on whether the samples had been taken from a ERBB2-overexpressing (9/25 vs 6/17; P=0.261, chi(2) test) or a non-ERBB2-overexpressing tumor (1/27 vs 0/8; P=0.581, chi(2) test). EGFR expression was only detected in 1/47 ERBB2-overexpressing primary tumors and 2/48 non-ERBB2-overexpressing tumors, and was undetectable in re-excised specimen. Taken together, we have demonstrated an increase in ERBB2 receptor activation in incompletely resected preinvasive breast cancer. We hypothesize that receptor phosphorylation is caused by growth factor stimulation in response to intraoperative tissue damage, and perioperative inhibition of specific cytokines could become a promising therapeutic strategy.

Purpose: This phase II study evaluated the efficacy and safety of ixabepilone as neoadjuvant therapy for invasive breast cancer not amenable to breast conservation surgery. Gene expression studies were undertaken using genes that were identified as potentially associated with sensitivity/resistance to ixabepilone in prior preclinical investigations., Patients and Methods: Patients with invasive breast cancer >or= 3 cm were eligible. Ixabepilone 40 mg/m(2) was administered as a 3-hour intravenous infusion on day 1 of a 21-day cycle for four or fewer cycles., Results: One hundred sixty-one patients were treated. The overall complete pathologic response (pCR) rate was 18% in breast and 29% in estrogen receptor (ER) -negative patients. Gene expression data were available for 134 patients. ER gene expression (ER1) was inversely related to pCR in breast and had a positive predictive value (PPV) of 37% and negative predictive value (NPV) of 92%. A 10-gene penalized logistic regression (PLR) model developed from 200 genes predictive of ixabepilone sensitivity in preclinical experiments included ER and tau and had higher PPV (45%) and comparable NPV (89%) to ER1. Grade 3 to 4 adverse events (AEs) were reported for 32% of patients. Except for neutropenia and leukopenia, all grade 3 to 4 AEs occurred in

Introduction: The role of the b-HCG/LH/LH-R system in breast cancer is conflicting. Whereas some reports suggest a protective effect of b-HCG on breast epithelium, vitro studies implicate a role of b-HCG/LH-R in the development and growth of breast tumors., Material and Methods: In order to further investigate a possible involvement of b-HCG/LH-R in breast carcinogenesis, immunofluorescence analyses of b-HCG/LH-R expression was performed on 70 preinvasive and adjacent invasive breast cancer specimen using tissue microarrays (TMAs)., Results: In 37 preinvasive samples available for further analysis, b-HCG/LH-R was found in 8/37 samples (21.6%; weak, intermediate and strong staining in 4/37 (10.8%), 2/37 (5.4%) and 2/37 (5.4%). In contrast, b-HCG/LH-R expression was observed in 19/27 (70.4%) adjacent invasive specimen with weak, moderate and strong immunostaining in 10/27 (37.0%), 6/27 (22.2%) and 3/27 (11.1%), respectively. This was statistically significant when compared to preinvasive components (P = 0.001, Chi Square Test)., Conclusions: Based on the observation that b-HCG/LH-R was found to be selectively upregulated in invasive tumor components, we suggest that under certain circumstances, sensitivity of ductal cells to hormones that target b-HCG/LH-R could favour mammary carcinogenesis.

Background: Vasomotor symptoms and bone loss are complications frequently induced by adjuvant treatment for breast cancer. Tibolone prevents both side-effects, but its effect on cancer recurrence is unknown. The aim of this study was to show non-inferiority of tibolone to placebo regarding risk of recurrence in breast-cancer patients with climacteric complaints., Methods: Between July 11, 2002, and Dec 20, 2004, women surgically treated for a histologically confirmed breast cancer (T(1-3)N(0-2)M(0)) with vasomotor symptoms were randomly assigned to either tibolone 2.5 mg daily or placebo at 245 centres in 31 countries. Randomisation was done by use of a centralised interactive voice response system, stratified by centre, with a block size of four. The primary endpoint was breast-cancer recurrence, including contralateral breast cancer, and was analysed in the intention-to-treat (ITT) and per-protocol populations; the margin for non-inferiority was set as a hazard ratio of 1.278. This study is registered with ClinicalTrials.gov, number NCT00408863., Findings: Of the 3148 women randomised, 3098 were included in the ITT analysis (1556 in the tibolone group and 1542 in the placebo group). Mean age at randomisation was 52.7 years (SD 7.3) and mean time since surgery was 2.1 years (SD 1.3). 1792 of 3098 (58%) women were node positive and 2185 of 3098 (71%) were oestrogen-receptor positive. At study entry, 2068 of 3098 (67%) women used tamoxifen and 202 of 3098 (6.5%) women used aromatase inhibitors. The mean daily number of hot flushes was 6.4 (SD 5.1). After a median follow-up of 3.1 years (range 0.01-4.99), 237 of 1556 (15.2%) women on tibolone had a cancer recurrence, compared with 165 of 1542 (10.7%) on placebo (HR 1.40 [95% CI 1.14-1.70]; p=0.001). Results in the per-protocol population were similar (209 of 1254 [16.7%] women in the tibolone group had a recurrence vs 138 of 1213 [11.4%] women in the placebo group; HR 1.44 [95% CI 1.16-1.79]; p=0.0009). Tibolone was not different from placebo with regard to other safety outcomes, such as mortality (72 patients vs 63 patients, respectively), cardiovascular events (14 vs 10, respectively), or gynaecological cancers (10 vs 10, respectively). Vasomotor symptoms and bone-mineral density improved significantly with tibolone, compared with placebo., Interpretation: Tibolone increases the risk of recurrence in breast cancer patients, while relieving vasomotor symptoms and preventing bone loss., Funding: Schering-Plough (formerly NV Organon, Oss, Netherlands).

Background: Breast cysts are the most common cause of benign breast masses. Simple breast cysts do not need further evaluation, but complex breast cysts require additional assessment due to the potential presence of malignancy. However, these complex cysts have rarely been examined and quantified according to the associated cancer detection rate. Our study is the first investigation to evaluate the malignancy rate of complex breast cysts identified by histopathological results., Methods: Imaging findings of complex cysts were correlated retrospectively with clinical and pathologic outcomes. We detected a malignancy rate of 14%. Sonographic criteria of a complex cyst such as thick cyst wall (P = .0006), lobulation (P = .01), and hyperechogenicity (P = .04) were predictive of neoplasm. Two or more criteria combined were associated with a 13.6-fold higher risk of malignancy (P <.0001)., Conclusion: Based on our results we reinforce the importance of adequate assessment of complex breast cysts to prevent a missed or delayed diagnosis of malignancy.

Purpose: Breast diseases in teenage girls are fortunately uncommon, with most presenting masses being benign. The aim of this study was to evaluate the histopathological results of breast lesions excised from adolescent females less than 19 years of age., Methods: The authors reviewed the medical and pathology records at the University Hospital of Vienna, Department of Obstetrics and Gynaecology, between 1993 and 2006, retrospectively. All data included the patient age, age of menarche, pregnancy, hormonal contraception, family history of breast cancer, size of the breast lesion and its histopathology following surgery., Results: Thirty-seven female patients with an average age of 16 years (ranging 12-18 years) were operated on for breast tumor and/or discharge. All tumors were palpable. Six patients had bilateral breast masses; thus, 43 breast lesions were evaluated following surgical excision. Surprisingly, breast cancer was found in two cases. Both patients were diagnosed with a noninvasive ductal carcinoma in situ (DCIS) within a fibroadenoma at the age of 16. These are the first reported cases of DCIS found in this young age group. As breast neoplasm was found in two cases, a malignancy rate of 4.7% was observed. The most common histologies were fibroadenoma (n=27) and fibrocystic disease (n=4)., Conclusion: The incidence of primary breast cancer in adolescent women is low. However, our experience shows the need for compulsory excision of all breast masses and highlighting the importance of histopathological evaluation of all breast tumors including adolescents.

During the past few years, the intensified detection of small (mammary) carcinomas causes an increase in the number of mammary cancers. Cancer of the mammary tissues has an almost individually unpredictable behavior and aggressiveness. Therefore, a better insight in the molecular biological defects, which are responsible for initiation and progressive aggressiveness of mammary cancer, is necessary. Proteomics are an alternative to identify proteins which initiate carcinogenesis and can be useful to predict cancer prognosis. Today, the most commonly used technique for large-scale protein identification in clinical samples is two-dimensional electrophoresis (2-DE) in combination with image analysis and MS. Using these techniques, qualitative and quantitative information can be achieved regarding protein forms and post-translational modifications. In the following article we review proteomic techniques that are now commonly used in order to elucidate the role of proteins in breast cancer.

Purpose: BRCA1 mutation carriers are at high risk for breast cancer (BC). The risk management strategy may include radiological investigations for early detection or prophylactic mastectomy (PM). For a mutation carrier, PM may be more significant than surveillance alone when pre-malignant and malignant changes occur increasingly in mastectomy specimens, given normal findings on radiological investigations. In the present study we retrospectively investigated the differences between histological findings in PM specimens of BRCA1 carriers and those of a control group., Methods: Twenty-four healthy and 28 affected carriers in the presence of normal preoperative radiological findings were included in the study. To compare the frequency of pre-malignant and malignant lesions in PM specimens, a control group matched for age and disease status was included. T-tests for independent samples and Wilcoxon's signed-rank test were used for comparison of groups., Results: The entire study group differed significantly from the control group (42.3 vs. 5.8%; P < 0.001) in terms of the occurrence of pre-malignant and malignant lesions. Both, the sub-group comparison of healthy mutation carriers as well as diseased carriers with their controls, showed a significant difference in terms of the occurrence of pre-malignant and malignant changes (45.8 vs. 0%; P = 0.002; 39.3 vs. 10.7%; P = 0.03). In PM specimens of mutation carriers, carcinomas were identified in 5.8% (3/52) and pre-malignant changes in 36.5% (19/52)., Conclusions: BRCA1 mutation carriers should be informed of the fact that pre-malignant and even malignant changes are frequently found in PM specimens despite normal radiological findings.

Background: The Austrian Breast and Colorectal Cancer Study Group trial-12 (ABCSG-12) bone substudy assesses zoledronic acid for preventing bone loss associated with adjuvant endocrine therapy and reports on long-term findings of bone-mineral density (BMD) during 3 years of treatment and 2 years after completing adjuvant treatment with or without zoledronic acid. The aim of this substudy is to gain insight into bone health in this setting., Methods: ABCSG-12 is a randomised, open-label, phase III, 4-arm trial comparing tamoxifen (20 mg/day orally) and goserelin (3.6 mg subcutaneously every 28 days) versus anastrozole (1 mg/day orally) and goserelin (3.6 mg subcutaneously every 28 days), both with or without zoledronic acid (4 mg intravenously every 6 months) for 3 years in premenopausal women with endocrine-responsive breast cancer. This prospective bone subprotocol measured BMD at 0, 6, 12, 36, and 60 months. The primary endpoint of the bone substudy (secondary endpoint in the main trial) was change in BMD at 12 months, assessed by dual-energy X-ray absorptiometry in assessable patients. Analyses were intention to treat. Statistical significance was assessed by t tests. The ABCSG-12 trial is registered on the ClinicalTrials.gov website, number NCT00295646., Findings: 404 patients were prospectively included in the bone substudy and randomly assigned to endocrine therapy alone (goserelin and anastrozole or goserelin and tamoxifen; n=199) or endocrine therapy concurrent with zoledronic acid (goserelin, anastrozole, and zoledronic acid or goserelin, tamoxifen, and zoledronic acid; n=205). After 3 years of treatment, endocrine therapy alone caused significant loss of BMD at the lumbar spine (-11.3%, mean difference -0.119 g/cm(2) [95% CI -0.146 to -0.091], p<0.0001) and trochanter (-7.3%, mean difference -0.053 g/cm(2) [-0.076 to -0.030], p<0.0001). In patients who did not receive zoledronic acid, anastrozole caused greater BMD loss than tamoxifen at 36 months at the lumbar spine (-13.6%, mean difference -0.141 g/cm(2) [-0.179 to -0.102] vs -9.0%, mean difference -0.095 g/cm(2) [-0.134 to -0.057], p<0.0001 for both). 2 years after the completion of treatment (median follow-up 60 months [range 15.5-96.6]), patients not receiving zoledronic acid still had decreased BMD at both sites compared with baseline (lumbar spine -6.3%, mean difference -0.067 g/cm(2) [-0.106 to -0.027], p=0.001; trochanter -4.1%, mean difference -0.03 g/cm(2) [-0.062 to 0.001], p=0.058). Patients who received zoledronic acid had stable BMD at 36 months (lumbar spine +0.4%, mean difference 0.004 g/cm(2) [-0.024 to 0.032]; trochanter +0.8%, mean difference 0.006 g/cm(2) [-0.018 to 0.028]) and increased BMD at 60 months at both sites (lumbar spine +4.0%, mean difference 0.039 g/cm(2) [0.005-0.075], p=0.02; trochanter +3.9%, mean difference 0.028 g/cm(2) [0.003-0.058], p=0.07) compared with baseline., Interpretation: Goserelin plus tamoxifen or anastrozole for 3 years without concomitant zoledronic acid caused significant bone loss. Although there was partial recovery 2 years after completing treatment, patients receiving endocrine therapy alone did not recover their baseline BMD levels. Concomitant zoledronic acid prevented bone loss during therapy and improved BMD at 5 years.

Background: Breast cancer is characterized by malignant transformation of epithelial cells, but stromal cells also play an important role in tumorigenesis. While tumor-derived fibroblasts display unique phenotypic properties, it is unclear whether they also represent are a specific subpopulation., Materials and Methods: Stromal fibroblasts deriving from malignant tissue of 10 women with invasive breast cancer, and from normal breast tissue of 10 women with benign breast disorders, were subjected to differential complementary DNA Microarray Analysis by using a 2,400 gene cDNA array. Individual gene expression pattern were confirmed by RT-PCR., Results: In a cDNA array that allows to analyze the differential gene expression of more than 2,400 genes, the mRNA expression of 135 genes were increased more than 2 fold in fibroblasts from malignant breast tumors. The majority of these genes encode tumor-promoting cytokines, transcription factors and cell-matrix associated proteins. The mRNA expression of 110 genes decreased to less than 0.5 fold. The remaining 2,155 genes were not significantly altered. RT-PCR performed on individual biopsies from breast cancer and normal breast tissues confirmed the validity of the pooled gene expression signature., Conclusion: Breast cancer-derived stromal fibroblasts show a distinctive gene expression pattern that differentiates them from normal breast stroma. Our observation of increased expression of tumor promotion-associated genes even in the absence of adjacent malignant epithelium suggests that tumor stroma is comprised of a fibroblastic subpopulation that provides for a microenvironment which supports tumor growth and invasion.

Background: Luteinizing hormone (LH) and human chorionic gonadotropin (HCG) target their receptor in gonadal and nongonadal cells to stimulate steroidogenesis and cell growth. The aim of the present study was to investigate the expression of HCG/LH-R in endometriosis to elucidate a possible impact of LH and HCG on this disease., Materials and Methods: Analysis of HCG/LH-R protein expression in 23 paired samples of ectopic and eutopic tissue of cycling women with endometriosis and in endometrial samples from 22 healthy controls was conducted via immunofluorescence. HCG and HCG/LH-R gene expression in endometriotic lesions was confirmed by reverse-transcriptase polymerase chain reaction., Results: In endometriotic implants, epithelial HCG/LH-R was found in 12/23 samples. No significant differences in HCG/LH-R levels were observed when compared with glands of uterine endometrium from the same patients or healthy controls. Messenger RNA transcripts for HCG were detected in all 12 samples, whereas HCG/LH-R mRNAs were observed in 10 of the 12 endometriotic lesions investigated., Conclusions: Although HCG/LH-R was not found to be selectively upregulated in endometriosis, the mere presence of HCG/LH-R in endometriotic tissue may suggest sensitivity of endometriosis to HCG and LH that target HCG/LH-R.

Purpose: Intratumoral estradiol levels in postmenopausal women with breast cancer are thought to be mainly regulated by the aromatase-mediated conversion from androgens and estrogen sulfotransferase (EST)-mediated reduction of bioavailability. While in invasive breast cancer (IBC) the role of both enzymes has been extensively studied and has led to the use of aromatase inhibitors as a key therapeutic strategy, comparably little is still known about their role in the local regulation of estradiol in ductal carcinoma in situ (DCIS)., Methods: We have performed immunohistochemistry to investigate the expression of aromatase and sulfotransferase in custom-made breast cancer tissue arrays containing 96 samples of pure DCIS and in 104 tumor biopsies which contain both, DCIS and invasive components., Results: We found that aromatase was equally detectable in epithelial components of both, DCIS and IBC (P = 0.884, Chi square test). However, stromal aromatase expression was significantly higher in IBC compared to adjacent DCIS components (P = 0.034, Chi square test). Whereas no significant difference was observed for epithelial aromatase expression in high versus non-high grade DCIS (P = 0.735 Chi square test), epithelial EST levels were found to be significantly down-regulated in high-grade DCIS compared to non-high grade cases (P = 0.042)., Conclusion: We have demonstrated the presence of both aromatase and EST in malignant epithelium and adjacent stromal fibroblasts in DCIS. Lower stromal aromatase expression in preinvasive breast cancer and lower EST levels in high-grade DCIS suggest that the net effect of intratumoral estradiol (E2)-modulating enzymes results in lower local E2 levels in earlier stages of breast tumorigenesis.

Estetrol (E 4 ) is a pregnancy-specific D-ring metabolite of estradiol (E 2 ) and estriol (E 3 ) produced by the human fetal liver and present in both male and female fetuses. In adults, female exposure is restricted to the gestational period. We report that E 4 , dose-dependently, prevents the growth of chemically induced (7,12-dimethylbenz(a)anthracene, DMBA) mammary tumors in female Sprague-Dawley rats and that E 4 has the potential to reduce the number and size of pre-existing mammary tumors. We performed two prevention studies and one intervention study. In the prevention studies, we investigated the effect of oral doses of E 4 over a dose range of 0.5-3.0 mg/kg. The intervention study used oral dose levels of 1, 3 and 10 mg/kg E 4 . The antiestrogen tamoxifen was used as reference compound in all three studies and ovariectomy and ethinylestradiol, at estrogenic doses pharmacologically equipotent to E 4 , acted as control treatments in the second prevention study and in the intervention study. Rats treated with DMBA develop estrogen-responsive breast tumors. This model has become the standard pharmacological model to investigate the effect of new compounds on breast tumors. When DMBA-induced rats were co-treated with E 4 for 8 weeks, this resulted in a dose-dependent reduction in the number and size of tumors, an effect that appeared equally effective as tamoxifen treatment or ovariectomy and was not seen with ethinylestradiol. When E 4 was administered to rats in which tumors had already developed, a significant decrease in the number and size of tumors was observed after 4 weeks. This decrease was dose-dependent, comparable to tamoxifen-treated animals, and at high dose levels E 4 was as effective as ovariectomy.

Background: Clinical trial data have shown that among breast cancer patients who were disease free after 5 years of adjuvant treatment with tamoxifen, further extended treatment with the nonsteroidal aromatase inhibitor letrozole reduces breast cancer recurrence. We examined the efficacy and tolerability of extended adjuvant therapy with another aromatase inhibitor, anastrozole, for 3 years among women who had completed 5 years of adjuvant therapy., Methods: Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 6a is an extension of ABCSG Trial 6, in which hormone receptor-positive postmenopausal patients received 5 years of adjuvant tamoxifen, with or without the aromatase inhibitor aminoglutethimide, for the first 2 years of therapy. For ABCSG Trial 6a, patients who were disease free at the end of Trial 6 were randomly assigned to receive either 3 years of anastrozole or no further treatment. Efficacy data were analyzed with the use of a Cox proportional hazards regression model with two-sided P values and Kaplan-Meier curves, and tolerability data were estimated using logistic regression analysis with odds ratios and 95% confidence intervals (CIs)., Results: ABCSG Trial 6a included 856 patients. At a median follow-up of 62.3 months, women who received anastrozole (n = 387) had a statistically significantly reduced risk of recurrence (locoregional recurrence, contralateral breast cancer, or distant metastasis) compared with women who received no further treatment (n = 469; hazard ratio = 0.62; 95% CI = 0.40 to 0.96, P = .031). Anastrozole was well tolerated, and no unexpected adverse events were reported., Conclusions: These data confirm the benefit of extending adjuvant tamoxifen therapy beyond 5 years with anastrozole compared with no further treatment. Further research is required to define the optimum length of extended adjuvant therapy and to investigate the possibility of tailoring this period to suit different disease types.

Endometriosis is an estrogen-dependent gynecological disease causing pelvic pain and infertility. Impaired estrogen metabolism is thought to play a pivotal role in the pathogenesis of the disease. While there is some information on factors involved in the synthesis of E2, information on E2-deactivating enzymes is still very limited. To elucidate the intracrinology of endometriotic tissues, the authors analyze the expression of aromatase and E2-inactivating estrogen sulfotransferase (EST) in paired biopsies obtained simultaneously from the endometrium and from endometrial lesions of each of 35 patients with peritoneal or ovarian endometriosis and in cycling endometria from 33 women without endometriosis. Protein localization was demonstrated by immunohistochemistry. Aromatase expression was found in endometriotic glands in 32 of 35 cases and was elevated in comparison to corresponding uterine endometria (25 of 35 cases, P = .021, chi(2) test). The difference was even more pronounced when uterine endometria from endometriosis patients were compared with that of healthy controls (8 of 33 cases, P < .001, chi(2) test). The EST levels were essentially unchanged. The elevated expression of aromatase in eutopic and ectopic endometrium from patients with endometriosis in the presence of comparable EST provides further evidence for unopposed local biosynthesis of estrogens in endometriosis.

BRCA1/2 mutations predispose to early onset breast and ovarian cancers. The phenotypic expression of mutant alleles, however, is thought to be modified by factors that are also involved in the pathogenesis of sporadic breast cancer. One such protein is IGF-I, one of the strongest mitogens to breast cancer cells in vitro. We have utilized immunohistochemistry to compare the intratumoral IGF-I and IGF-I receptor (IGF-IR) protein expression in 57 BRCA1/2 mutation carriers and 102 matched breast cancer patients without a family history in a nested case-control study. BRCA1 silencing by siRNA was used to investigate the effect of BRCA mutations on IGF-I protein expression. IGF-I protein expression was detected in tumoral epithelium and surrounding stroma, and was significantly upregulated in tumors of BRCA mutation carriers when compared with matched sporadic tumors (epithelial: 87.7% vs 61.8%, P=0.001; stromal: 73.7% vs 34.3%, P<0.001). By contrast, IGF-IR protein expression was confined to malignant epithelium and was unchanged in mutation carriers (52.6% vs 39.2%, P=0.310). While in mutation carriers IGF-IR protein expression was significantly correlated with both epithelial (P=0.003) and stromal IGF-I (P=0.02), this association was less pronounced in sporadic breast cancer (P=0.02 respectively). siRNA-mediated downregulation of BRCA1 in primary human mammary gland cells triggered upregulation of endogenous intracellular IGF-I in vitro. The increased intratumoral IGF-I protein expression in BRCA mutation carriers suggests an involvement of the IGF-I/IGF-IR axis in the biological behavior of breast cancers in this population and could define a potential therapeutic target.

Many patients with a history of breast cancer (BC) will suffer from vasomotor symptoms, which can be induced or exacerbated by treatment with tamoxifen or aromatase inhibitors. The LIBERATE trial was designed as a randomized, double-blind, multicenter trial to demonstrate that tibolone 2.5mg/day (Livial) is non-inferior to placebo regarding BC recurrence in women with vasomotor symptoms surgically treated for primary BC within the last 5 years. Secondary objectives are effects on vasomotor symptoms as well as overall survival, bone mineral density and health-related quality of life. Mean age at randomization was 52.6 years, and the mean time since surgery was 2.1 years. The mean daily number of hot flushes and sweating episodes was 7.3 and 6.1, respectively. For the primary tumor, Stage IIA or higher was reported for >70% of the patients. In subjects whose receptor status was known, 78.2% of the tumors were estrogen receptors positive. At randomization, tamoxifen was given to 66.2% of all patients and aromatase inhibitors to 7%. Chemotherapy was reported by 5% at randomization. The adjuvant tamoxifen use in LIBERATE allows a comparison with the Stockholm trial (showing no risk of BC recurrence associated with hormone therapy), which was stopped prematurely subsequent to HABITS. The LIBERATE trial is the largest, ongoing, well-controlled study for treatment of vasomotor symptoms in BC patients.

Neoadjuvant chemotherapy is the treatment of choice for locally-advanced breast cancer and leads to down staging and improved breast-conserving therapy (BCT) rates. While its efficacy is well established, considerably less is known about the most effective regimen. We have performed a retrospective analysis of 132 breast cancer patients who had undergone neoadjuvant chemotherapy at our institution. Patients had either received a) anthracyclines ("A", n=35), b) anthracyclines and taxanes ("AT", n=55), or c) neither of the two compounds ("NoA/T", n=42). Clinical response, pathological response and survival were evaluated in each arm. While all three regimens resulted in significant tumor regression, AT was most effective with a mean tumor shrinkage of 39% (ultrasound) and 41% (mammography) (Kruskal-Wallis, p=0.004, and p=0.027). Breast conservation was achieved in 75% by AT, in 49% by A, and in 19% by NoA/T (Kruskal-Wallis, p<0.001). The treatment groups did not differ in respect to pathological complete response (pCR) (chi2-test, p=0.068), although higher cumulative anthracycline doses were predictive of pCR in multivariate analyses (p=0.022). While the mammographic but not the ultrasound-determined tumor diameter determined whether a woman underwent BCT, only an ultrasound-determined size reduction was predictive for disease-free survival (DFS) and overall survival (OS) (log rank, p=0.0093, and p=0.044, respectively). Other parameters that affected BCT rates were age (p= 0.003), year of diagnosis (p=<0.001), presence of multifocal disease (p= 0.032) and the cumulative anthracycline dose (p= <0.001). While the combination of anthracyclines and taxanes is most effective in achieving clinical remission and BCT, the cumulative anthracycline dose appears most important for achieving pCR.

Overexpression of HER family members is a well established prognostic factor and identifies potential targets for antibody-based receptor blocking strategies. While several studies have analyzed the expression of HER2 and other HER-family members in malignant tumors, considerably less is known about their expression and activation in non-involved breast tissue from breast cancer patients. We have therefore investigated the differential expression of EGFR, HER2, and their tyrosine-kinase activated forms (ptyr-1248 Her-2 and ptyr-845 EGFR) in 63 tumor specimen containing: a) malignant epithelium, b) in non-malignant tissue located at the peritumoral margin, and c) in uninvolved breast tissue obtained from tissue distant from the tumor. Using immunohistochemistry (IHC), we found significantly higher HER2 protein expression levels in malignant epithelium than in marginal and peripheral non-malignant epithelium (p=1.3 x 10(-10) Fisher's exact test). Epithelial EGFR expression did not differ between the three tissue types, but stromal EGFR protein was significantly more common in marginal and peripheral tissues when compared to tumor tissues (p=0.008, Fisher's exact test). When analyzing activated receptor forms, we found epithelial ptyr-1248 HER2 expression in one tumoral, one marginal and one peripheral sample. We did not observe ptyr-845 EGFR in any of the samples analyzed. We found a significant overall correlation between epithelial and stromal EGFR expression (r=0.442; p<0.0001; Spearman's Rho), and between stromal EGFR expression and normal tissue type (r=0.170; p<0.02; Spearman's Rho). Epithelial HER2 expression and normal tissue type (r=0.492; p<0.0001; Spearman's Rho) were inversely correlated. Taken together, we have observed a differential expression pattern of EGFR, HER2, and activated HER2 that is dependent on the spatial relation to a malignant tumor. Our findings of decreased intratumoral EGFR expression and the absence of activated EGFR suggests that, in contrast to HER2, EGFR inhibition might not be an ideal target for antibody therapy.

Purpose: Tibolone is a selective tissue estrogenic activity regulator, approved for the treatment of vasomotor symptoms in postmenopausal women. We have done an exploratory, double-blind, randomized, placebo-controlled pilot trial to investigate the tissue-specific effects of 2.5 mg tibolone on breast cancer in postmenopausal women, in particular on tissue proliferation (STEM, Study of Tibolone Effects on Mamma carcinoma tissue)., Experimental Design: Postmenopausal women with initially stage I/II, estrogen receptor-positive (ER+) primary breast cancer, were randomly assigned to 14 days of placebo or 2.5 mg/d tibolone. Core biopsies of the primary tumor were obtained before and after treatment. Ki-67 and apoptosis index were analyzed in baseline and corresponding posttreatment specimen., Results: Of 102 enrolled patients, 95 had evaluable data. Baseline characteristics were comparable between both treatment groups. Breast cancer cases are mainly invasive (99%), stage I or II (42% and 50% respectively), and ER+ (99%). Median intratumoral Ki-67 expression at baseline was 13.0% in the tibolone group and 17.8% in the placebo group, and decreased to 12.0% after 14 days of tibolone while increasing to 19.0% in the placebo group. This change from baseline was not significantly different between tibolone and placebo (Wilcoxon test; P=0.17). A significant difference was observed between the treatment groups when the median change from baseline apoptosis index was compared between the treatment groups (tibolone, 0.0%; placebo, +0.3%; Wilcoxon test; P=0.031). The incidence of adverse effects was comparable., Conclusions: In ER+ breast tumors, 2.5 mg/d tibolone given for 14 days has no significant effect on tumor cell proliferation.

Purpose: Preoperative (neoadjuvant) chemotherapy for operable breast cancer downstages tumors initially not suitable for breast-conserving surgery. A pathologic complete response (pCR) to neoadjuvant chemotherapy may be a surrogate for longer overall survival, but this beneficial effect remains to be established. This phase III trial evaluated whether doubling the number of cycles of neoadjuvant treatment increased the pCR rate., Patients and Methods: Patients with biopsy-proven breast cancer (T1-4a-c, N+/-, M0; stage I to III) were eligible and randomly assigned to either three or six cycles of epirubicin 75 mg/m2 and docetaxel 75 mg/m2 on day 1 and granulocyte colony-stimulating factor on days 3 through 10 (ED+G), every 21 days. The primary end point was the pCR rate of the breast tumor. Secondary end points were pathologic nodal status after surgery and the rate of breast-conserving surgery., Results: A total of 292 patients were accrued, and 288 patients were assessable for efficacy and safety. Groups were well balanced for known prognostic factors. Six cycles of ED+G, compared with three cycles, resulted in a significantly higher pCR rate (18.6% v 7.7%, respectively; P = .0045), a higher percentage of patients with negative axillary status (56.6% v 42.8%, respectively; P = .02), and a trend towards more breast-conserving surgery (75.9% v 66.9%, respectively; P = .10). Rates of adverse events were similar, and no patients died on treatment., Conclusion: Doubling the number of neoadjuvant ED+G cycles from three to six results in higher rates of pCR and negative axillary nodal status with no excess of adverse effects. Thus, six cycles of ED+G should be the standard neoadjuvant treatment for operable breast cancer if this combination is chosen.

The suppression of local estrogens levels is of key importance in the treatment of ER-positive breast cancer. Essentially all endocrine strategies act by either suppressing estrogen formation or competitively inhibiting receptor-binding in tumor cells. Nevertheless, little is still known about the local expression of aromatase and sulfotransferase which are the key modulators of intra-tumoral estrogen levels. We have performed immunohistochemostry to investigate the expression of aromatase and sulfotransferase in 42 samples obtained directly from malignant breast tumors, and compared it to biopsies obtained from uninvolved tissue in the vicinity of the invasion front, and to distant breast tissue. We found that aromatase was equally detectable in both tumor epithelial and stroma, but was mostly epithelial in non-malignant tissues (P=0.00008, Fisher's exact test). Also, aromatase protein expression was significantly more common in tumoral stroma when compared with peritumoral and distant breast stroma (P=0.00005, and P<0.00001 respectively). With the notable exception of cystosarcoma phylloides, sulfotransferase protein was detectable only in epithelial tissues, regardless of the location within the diseased breast. However, epithelial sulfotransferase was correlated with epithelial aromatase (r=0.35461, P=0.0009, Spearman's rho test) and with the epithelial ER status (r=0.29313, P=0.005). We have demonstrated a differential aromatase and sulfotransferase protein expression pattern that is dependent on the spatial relation to a malignant breast tumor. Our results indicate a net increase in intratumoral active estrogen levels through increased stromal aromatization, while physiological local inactivation by sulfotransferase activity remains essentially unchanged.

Her-2/neu overexpression in human breast cancer leads to an aggressive biological behavior and poor prognosis. Although the anti-Her-2/neu antibody trastuzumab (Herceptin(R)) has become a valuable therapeutic option for patients with Her-2/neu-overexpressing breast cancer, many patients do not benefit from this therapy. To evaluate the effect of receptor activation on tumor response, we have investigated the phosphorylation status of Her-2/neu and EGFR in 46 Her-2/neu-overexpressing tumor samples from trastuzumab-treated metastatic breast cancer patients by immunohistochemistry. Activated (p)tyr-1248 Her-2/neu was detected in 9 of 46 breast cancers (20%), and activated (p)tyr-845 and (p)tyr-1173 EGFR were both present in 6 tumors (13%) while EGFR was present in 16 cases (35%). ptyr-1248 Her-2/neu showed a trend to correlate with increased response to trastuzumab (p = 0.063), while ptyr-845, ptyr-1173 EGFR and EGFR did not. The presence of ptyr-1248 Her-2/neu and ptyr-845 or ptyr-1173 EGFR, however, was a strong predictor of both response to trastuzumab-based treatment (OR = 8.0, p = 0.021 and OR = 8.0, p = 0.021) and clinical benefit (OR = 5.47, p = 0.041 and OR = 6.22, p = 0.028 multivariate logistic regression analysis). Furthermore, ptyr-845 EGFR and ptyr-1248 Her-2/neu were both independent predictors of progression-free survival (RR = 0.21, p = 0.01 and RR = 0.45, p = 0.026, multivariate analysis). Patients with ptyr-845 EGFR positive tumors also tended toward increased overall survival (RR = 0.17, p = 0.082). Taken together, we have demonstrated that the determination of activated EGFR improves the utility of ptyr-1248 Her-2/neu staining in predicting the clinical outcome of patients undergoing trastuzumab treatment. We hypothesize that the activation state of both Her-2/neu and EGFR are key determinants for trastuzumab efficacy., ((c) 2005 Wiley-Liss, Inc.)

Gene expression analysis has become a promising tool in predicting the clinical course of malignant disease and the response to antineoplastic therapy. Surprisingly, only little is known about the protein expression pattern of human tumors. Recent advances in proteomic analysis allow proteins of interest to be identified by their expression and/or modification pattern in 2-DE rather than using the traditional approach of translating gene expression data. To identify a proteomic pattern that is characteristic for malignant breast epithelium, we performed differential 2-DE analysis in sets of microdissected malignant breast epithelia and corresponding adjacent normal breast epithelia from five patients with invasive breast carcinoma. Thirty-two protein spots were found to be selectively regulated in malignant epithelium, and were subjected to MALDI-TOF and/or immunoblotting for protein identification. Thirteen of the identified proteins had previously not been associated with breast cancer. The validity of these findings was confirmed by literature review and immunohistochemistry for identified proteins in an independent cohort of 50 breast cancer specimens. We here describe, for the first time, a proteomic analysis of matched normal and malignant epithelia from invasive breast carcinomas. This strategy leads to a better understanding of oncogenesis at an operational level and helps to characterize the malignant phenotype of individual tumors, and thereby to identify novel targets for antineoplastic therapy.

CrkL is a nuclear adaptor and transcriptional activator in Bcr-Abl expressing cells and constitutes the major tyrosine phosphorylated protein in CML, but the expression and biological function of CrkL in other malignancies is largely unknown. Using immunohistochemistry, we have analyzed the protein expression of activated (p)CrkL in normal and malignant tissues. We then treated K562 leukemia cells with imatinib to analyze the effect of tyrosine kinase inhibition on CrkL activation. pCrkL expression was predominantly epithelial and detected in the majority of non-malignant prostate (79%), 49% of colon biopsies, 36% of skin biopsies, and 41% of samples obtained from normal brain. Protein expression was, however, considerably less frequent in normal breast (18%), lung (16%) and ovarian (12%) tissues. In contrast to their corresponding benign tissues, pCrkL expression was significantly more common in breast cancer samples (49%, p<0.0001; Fisher's exact test), lung carcinomas (55%, p=0.0002), lymphatic tissues (80% vs. 10%, p=0.012), skin cancer (67%, p=0.020), ovarian malignomas (50%, p<0.0001) and colon carcinomas (63%, p<0.03). By contrast, activated CrkL was significantly less frequent in prostate carcinoma samples when compared to corresponding non-malignant prostatic tissues (14% vs. 79%, p<0.0001). pCrkL expression was abrogated in K562 cells with the addition of the tyrosine kinase inhibitor imatinib, which indicates that phosphorylation of CrkL is mediated through targets of therapeutic TK inhibition. We hypothesize that pCrkL is selectively up-regulated in a number of malignant tumor entities and involved in malignant transformation. We further suggest that pCrkL might serve as a potential surrogate parameter for the efficacy of therapeutic TK inhibition.

While interleukins (IL)-1alpha and beta are thought to play an important role in malignant disease, little is still known about their expression in breast cancer. We have used reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) to analyze the expression of IL-1alpha and beta in breast cancer tissues, and compared their expression to estrogen receptor (ER) status and grading. In breast cancer cell lines, we found an inverse correlation between IL-1alpha and beta gene expression and differentiation, and only one highly invasive tumor cell line expressed IL-1alpha protein, while IL-beta was not detectable. Breast cancer tissue expressed variable amounts of IL-1alpha and beta messenger RNA, but consistently high levels of IL-1 type I receptor. IL-1alpha protein was detectable in malignant epithelium and adjacent stroma in 88% of cases. IL-1alpha expression was correlated with poor differentiation (P= 0.002; r= 0.469) and decreasing ERalpha expression (P= 0.004; r=-0.387). Stromal IL-1alpha was confined to areas with low or absent ERalpha protein expression in adjacent tumor epithelium (P= 0.001; r=-0.457). Taken together, we have demonstrated a functional IL-1 system in breast cancer and observed an inverse correlation between IL-1alpha and sex steroid receptor expression. We suggest that the expression of IL-1alpha in poorly differentiated, ERalpha-negative tumors contributes to their malignant phenotype.

Transglutaminase-2 is involved in the physiological regulation of cell growth, but has also been associated with a number of cancer-associated features such as cell adhesion, metastasis and extracellular matrix modulation. Despite its importance in tumor cell progression and survival, relatively little is known about its expression in human malignancies. We have therefore investigated the transglutaminase-2 expression pattern in breast and ovarian cancer by using tissue arrays which contained 57 invasive breast cancer biopsies and 62 ovarian cancers, and compared it to transglutaminase-2 protein levels in normal human tissues. By using immunohistochemistry, transglutaminase-2 protein was detected in 48 of 57 breast tumors (84%), with epithelial expression in 26 of 41 (63%) ductal invasive carcinomas and in all 6 (100%) lobular invasive carcinomas. Stromal transglutaminase-2 was present in 14 of 41 (34%) ductal subtypes and in 4 of 6 (67%) lobular subtypes, which is in sharp contrast to the infrequent expression in normal breast stroma (P<0.001, Mann-Whitney test) and somewhat also in normal breast epithelium (P = 0.065, Mann-Whitney test). In most other human tissues, transglutaminase-2 protein was less frequent and usually confined to either the epithelium or in adjacent stroma. In ovarian tumors, the protein was detected in 36 of the 62 cases (58%), and seen in all histological subtypes. Taken together, we have demonstrated increased transglutaminase-2 protein expression in both malignant breast epithelium and surrounding stroma, although its selective spatial expression pattern in normal tissues also indicates a physiological role in stromal-epithelial interactions.

Objectives: The activator protein-1 (AP-1) is a dimeric transcription factor formed by members of the Jun and Fos protein family. AP-1 plays a role in a variety of physiological functions including cell proliferation and differentiation, although both c-Jun and c-Fos have also been implicated in oncogenic transformation and tumor progression. To further elucidate the role of AP-1 in breast cancer, we have investigated the expression of the AP-1 proteins c-Jun, JunB, JunD, phosphorylated c-Jun, c-Fos, Fral, Fra2 and the tumor supressor protein p53., Methods: Protein expression was evaluated on a breast cancer tissue microarray with 58 lymph node positive or negative breast cancer specimens, 29 corresponding lymph node metastases, and 11 tissue samples from surrounding tumor-free tissue, each cored as triplicate. Jun and Fos protein family expression was evaluated by immunohistochemistry and was correlated with clinicopathological parameters., Results: High expression levels were observed for c-Jun, JunD, c-Fos and Fra2, whereas JunB and Fral exhibited lower staining. c-Jun protein expression was correlated to Fral staining (p = 0.007, Kendall's Tau) and Fral was further associated with c-Fos (p < 0.001), JunD (p = 0.001) and Fra2 (p = 0.011) expression. JunD expression correlated with c-Fos (p < 0.001), JunB (p = 0.035) and c-Jun (p = 0.05). Activated c-Jun correlated with c-Fos expression (p = 0.041). JunB was negatively correlated to tumor stage, (p = 0.093, corr coeff. = -0.293, Spearman's correlation) but was significantly increased in nodal negative tumors (p = 0.004, Mann Whitney test). In addition, increased Fral expression showed a trend towards an increased overall survival (p = 0.077, RR = 0.534, Cox regression)., Conclusion: Our results suggest an important role for JunB and Fral in the biological behavior of malignant breast tumors.

The antibody trastuzumab inhibits signal transduction in Her-2/neu overexpressing human breast cancer. However, the activation of co-expressed EGFR has also been show to additionally modulate the anti-tumoural effects of this drug. Similar to Her-2/neu, the extra cellular binding region of EGFR is believed to be proteolytically released from the cell surface upon receptor activation and can be detected in patients' serum (sEGFR). Considering the biological significance of an interaction between EGFR and Her-2/neu signalling in other human malignancies, we have investigated if trastuzumab treatment would affect sEGFR in 33 patients with Her-2/neu overexpressing metastatic breast cancer. We detected EGFR expression in 33% of Her-2/neu overexpressing breast tumours. In contrast to serum Her-2/neu (ECD) levels, which were correlated with the degree of Her-2/neu expression (P=0.048, Mann-Whitney test), we did not detect significant differences between sEGFR serum levels in EGFR expressing or non-expressing tumours. Furthermore, sEGFR serum levels were not correlated with clinical parameters such as response or clinical benefit rates, and no association was found between increased sEGFR levels and progression-free survival or overall survival. While we have previously observed a selective and significant decrease of ECD levels in patients who derived a clinical benefit from trastuzumab treatment during the first weeks of treatment, we were unable to find similar alterations in sEGFR concentrations. We therefore conclude that the measurement of systemic sEGFR levels in addition to ECD serum concentrations do not allow the prediction of clinical course of trastuzumab-treated patients more accurately.

Mutations in the BRCA1 gene result in an elevated risk of breast cancer (BC) and ovarian cancer (OC). However, risk estimates vary depending on the study population and statistical methodology used, and there are indications that the birth cohort and location of the mutation influence cancer risk. We investigated the risks for BC and OC associated with BRCA1 mutations in a young cohort of female mutation carriers who were identified by molecular genetic testing and belonged to a genetically heterogeneous Central European population. The study included 106 healthy and 158 affected carriers identified at an Austrian risk evaluation center. Risk estimation employed the product limit method. The log rank test was used to compare different strata. The risk of developing cancer to age 70 was found to be 85% for BC (95% CI 75-97%) and 53% for OC (95% CI 37-68%). Female mutation carriers born in 1958 or later were subject to a significantly higher risk of BC (P=0.005; 27% vs. 46% to age 40) and OC (P=0.006; 2% vs. 8% to age 40) than those born earlier. Mutations in exon 11 were associated with lower BC risk than mutations in exons 1-10 (P=0.008) and exons 12-24 (P=0.0006). OC risk was not influenced by mutation location (P=0.86). We conclude that female BRCA1 mutation carriers should be counseled about their cohort-dependent cancer risk. Further research into variables that affect cancer risk and are amenable to modification (e.g., lifestyle-related factors) should be considered a priority., (Copyright 2005 Wiley-Liss, Inc.)

Objective: To evaluate the local matrix metalloproteinase 1 (MMP-1), estrogen receptor (ER) alpha, and ERbeta protein expression in eutopic and dystopic tissue from patients with endometriosis and to compare the endometrial expression pattern in women with and without endometriosis., Design: Immunohistochemical analysis of MMP-1, ERalpha, and ERbeta in paired samples of uterine and endometriotic endometrium from cycling women with endometriosis and in endometrial tissue from 37 healthy women., Setting: Research laboratory at a medical school., Patient(s): Thirty-seven matched samples from endometriotic and corresponding endometrial biopsies obtained during the proliferative and secretory phase. Thirty-seven endometrial biopsies obtained from healthy women during comparable cycle phases., Intervention(s): Sampling of endometrial and endometriotic tissue., Main Outcome Measure(s): Matrix metalloproteinase 1, ERalpha, and ERbeta protein expression in paraffin-embedded tissue biopsies was measured by IHC., Result(s): In patients with endometriosis, epithelial and stromal cells from endometriotic lesions both express significantly higher levels of MMP-1 and lower levels of ERalpha than corresponding cells in uterine endometrium. Endometriotic epithelium also expresses higher levels of ERbeta than of ERalpha. In both endometrial glands and corresponding endometriotic epithelium, the distribution of MMP-1 is correlated with ERbeta. No significant differences in endometrial ERalpha, ERbeta, or MMP-1 expression could, however, be detected when patients with endometriosis and healthy controls were compared., Conclusion(s): We have shown that MMP-1 and ERbeta are coexpressed and up-regulated in endometriotic lesions, whereas local ERalpha expression is down-regulated. The altered ERbeta/ERalpha ratio in endometriotic glands suggests that estrogenic effects on MMP-1 are primarily mediated via ERbeta, and the local control of MMP-1 in eutopic endometrium is not different from that observed in healthy cycling women.