Deregulated ephrin-B2 signaling in mammary epithelial cells alters the stem cell compartment and interferes with the epithelial differentiation pathway

Cancer most probably originates from stem/progenitor cells and exhibits a similar cell hierarchy as normal tissues. Moreover, there is growing evidence that only the stem cells are capable of metastasis formation. We have previously shown that overexpression of a dominant negative ephrin-B2 mutant interferes with mammary gland differentiation and confers a metastatic phenotype to NeuT-induced mammary tumors with an increase in cells with stem/progenitor characteristics. To investigate the role of ephrin-B2 in the control of the mammary stem cell niche, we analyzed the mammary stem and progenitor cell populations in transgenic mice overexpressing the mutant ephrin-B2. Quantification by FACS analysis revealed a significant increase of cells in the basal/alveolar cell-, the bi-potent progenitor- and the stem cell-enriched fractions. Moreover, the supposed precursors of estrogen receptor-positive cells were elevated in the stem cell-enriched fraction. In contrast, the epithelium from transgenic mice overexpressing the native ephrin-B2 gene showed an augmentation of the luminal cell- and the bi-potent progenitor-enriched fractions. Repopulation assays revealed that the epithelial cells of truncated ephrin-B2 transgenic epithelial cells have a higher regeneration capacity than those of controls and of native ephrin-B2 transgenic mice, confirming the augmentation of stem cells. Morphologically, these outgrowths exhibited impaired basal/luminal compartmentalization and epithelial polarization. These results demonstrate that deregulated ephrin-B2 expression interferes with the regulation of the stem cell niche and leads to a shift of the differentiation pathway and may thereby contribute to the acquisition of the metastatic phenotype long before carcinogenic growth becomes apparent.

[Sarcomas and mixed mesodermal tumors of the uterus]

Malignant mesodermal tumors of the uterus are an inhomogenous group of uterine malignancies with different pathogenesis, clinical presentation and prognosis. These rare tumors represent approximately 1 % of all uterine malignancies. The aggressive carcinosarcomas or mixed muellerian tumors are defined by mixed malignant epithelial and malignant mesodermal histopathology and are of the same precursor cell origin like endometrial cancer. Thus, carcinosarcomas were reclassified by the FIGO as an aggressive type of endometrial cancer and treated like type II endometrial cancer. Adenosarcomas are also mixed tumors with benign epithelial proliferation and malignant mesodermal cell growth, have a good prognosis and represent less than 5 % of all mesodermal uterine malignancies. Besides carcinosarcomas, the pure mesodermal leiomyosarcomas are the most common mesodermal malignancies. Patients with leiomyosarcamos are usually perimenopausal, and although more than half of the patients present with symptoms, diagnosis occurs incidentally in most cases in final histopathologic workup of an excised putative myoma or uterus. Adequate anamnesis, gynecologic examination and careful imaging by transvaginal ultrasound in the preoperative setting might hint to correct differential diagnosis in many cases. Overall the prognosis of uterine leiomyomas is poor. Malignancies of the endometrial stroma are very rare and divided in two subgroups, the mostly estrogen receptor positive endometrial stromal sarcoma, which occur preferably in premenopausal women and show a favorable prognosis, and the very aggressive undifferentiated endometrial sarcomas. The more rare undifferentiated endometrial sarcomas occur in postmenopausal women and most patients die in the first two years after diagnosis. Risk stratification of preoperative differential diagnosis requires improvements and the correct histopathologic workup of mesodermal uterine malignancies is still a challenge for pathologists.

Recommendations for raloxifene use in daily clinical practice in the Swiss setting

Raloxifene is the first selective estrogen receptor modulator that has been approved for the treatment and prevention of osteoporosis in postmenopausal women in Europe and in the US. Although raloxifene reduces the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer, it is approved in that indication in the US but not in the EU. The aim was to characterize the clinical profiles of postmenopausal women expected to benefit most from therapy with raloxifene based on published scientific evidence to date.

17Beta-estradiol affects the response of complement components and survival of rainbow trout (Oncorhynchus mykiss) challenged by bacterial infection

Research on the endocrine role of estrogens has focused on the reproductive system, while other potential target systems have been less studied. Here, we investigated the possible immunomodulating role of 17beta-estradiol (E2) using rainbow trout (Oncorhynchus mykiss) as a model. The aims of the study were to examine a) whether estrogens can modulate immune gene transcription levels, and b) whether this has functional implications for the resistance of trout towards pathogens. Trout were reared from fertilization until 6 months of age under (1) control conditions, (2) short-term E2-treatment (6-month-old juveniles were fed a diet containing 20 mg E2/kg for 2 weeks), or c) long-term E2-treatment (twice a 2-h-bath-exposure of trout embryos to 400 mug 17beta-estradiol (E2)/L, followed by rearing on the E2-spiked diet from start-feeding until 6 months of age). Analysis of plasma estrogen levels indicated that the internal estrogen concentrations of E2-exposed fish were within the physiological range and analysis of hepatic vitellogenin mRNA levels indicated that the E2 administration was effective in activating the endogenous estrogen receptor pathway. However, expression levels of the hepatic complement components C3-1, C3-3, and Factor H were not affected by E2-treatment. In a next step, 6-month-old juveniles were challenged with pathogenic bacteria (Yersinia ruckeri). In control fish, this bacterial infection resulted in significant up-regulation of the mRNA levels of hepatic complement genes (C3-1, C3-3, Factor B, Factor H), while E2-treated fish showed no or significantly lower up-regulation of the complement gene transcription levels. Apparently, the E2-treated trout had a lower capacity to activate their immune system to defend against the bacterial infection. This interpretation is corroborated by the finding that survival of E2-treated fish under bacterial challenge was significantly lower than in the control group. In conclusion, the results from this study suggest that estrogens are able to modulate immune parameters of trout with functional consequences on their ability to cope with pathogens.

Rumen-protected choline supplementation in periparturient dairy goats: effects on liver and mammary gland

The current study investigated the effects of supplementing rumen-protected choline (RPC) on metabolic profile, selected liver constituents and transcript levels of selected enzymes, transcription factors and nuclear receptors involved in mammary lipid metabolism in dairy goats. Eight healthy lactating goats were studied: four received no choline supplementation (CTR group) and four received 4g RPC chloride/day (RPC group). The treatment was administered individually starting 4 weeks before expected kidding and continuing for 4 weeks after parturition. In the first month of lactation, milk yield and composition were measured weekly. On days 7, 14, 21 and 27 of lactation, blood samples were collected and analysed for glucose, beta-hydroxybutyrate, non-esterified fatty acids and cholesterol. On day 28 of lactation, samples of liver and mammary gland tissue were obtained. Liver tissue was analysed for total lipid and DNA content; mammary tissue was analysed for transcripts of lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory binding proteins 1 and 2, peroxisome proliferator-activated receptor gamma and liver X receptor alpha. Milk yield was very similar in the two groups, but R PC goats had lower (P < 0.05) plasma beta-hydroxybutyrate. The total lipid content of liver was unaffected (P = 0.890), but the total lipid/DNA ratio was lower (both P < 0.05) in RPC than CTR animals. Choline had no effect on the expression of the mammary gland transcripts involved in lipid metabolism. The current plasma and liver data indicate that choline has a positive effect on liver lipid metabolism, whereas it appears to have little effect on transcript levels in mammary gland of various proteins involved in lipid metabolism. Nevertheless, the current results were obtained from a limited number of animals, and choline requirement and function in lactating dairy ruminants deserve further investigation.

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

Background Clinical relevance of tumor infiltrating lymphocytes (TILs) in breast cancer is controversial. Here, we used a tumor microarray including a large series of ductal and lobular breast cancers with long term follow up data, to analyze clinical impact of TIL expressing specific phenotypes and distribution of TILs within different tumor compartments and in different histological subtypes. Methods A tissue microarray (TMA) including 894 ductal and 164 lobular breast cancers was stained with antibodies recognizing CD4, FOXP3, and IL-17 by standard immunohistochemical techniques. Lymphocyte counts were correlated with clinico-pathological parameters and survival. Results CD4+ lymphocytes were more prevalent than FOXP3+ TILs whereas IL-17+ TILs were rare. Increased numbers of total CD4+ and FOXP3+ TIL were observed in ductal, as compared with lobular carcinomas. High grade (G3) and estrogen receptor (ER) negative ductal carcinomas displayed significantly (p < 0.001) higher CD4+ and FOXP3+ lymphocyte infiltration while her2/neu over-expression in ductal carcinomas was significantly (p < 0.001) associated with higher FOXP3+ TIL counts. In contrast, lymphocyte infiltration was not linked to any clinico-pathological parameters in lobular cancers. In univariate but not in multivariate analysis CD4+ infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3+/CD4+ ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033). Conclusions Ductal and lobular breast cancers appear to be infiltrated by different lymphocyte subpopulations. In ductal cancers increased CD4+ and FOXP3+ TIL numbers are associated with more aggressive tumor features. In survival analysis, absolute numbers of TILs do not represent major prognostic indicators in ductal and lobular breast cancer. Remarkably however, a ratio > 1 of total FOXP3+/CD4+ TILs in ductal carcinoma appears to represent an independent favorable prognostic factor.

ImmunoChip study implicates antigen presentation to T cells in narcolepsy

Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin) deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.

In vitro metacestodicidal activities of genistein and other isoflavones against Echinococcus multilocularis and Echinococcus granulosus

Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.

Tamoxifen after adjuvant chemotherapy for premenopausal women with lymph node-positive breast cancer: International Breast Cancer Study Group Trial 13-93

PURPOSE: The value of adjuvant tamoxifen after chemotherapy for premenopausal women with breast cancer has not been adequately assessed. PATIENTS AND METHODS: Between 1993 and 1999, International Breast Cancer Study Group Trial 13-93 enrolled 1,246 assessable premenopausal women with axillary node-positive, operable breast cancer. All patients received chemotherapy (cyclophosphamide plus either doxorubicin or epirubicin for four courses followed by immediate or delayed classical cyclophosphamide, methotrexate, and fluorouracil for three courses), which was followed by either tamoxifen (20 mg daily) for 5 years or no further treatment. The primary end point was disease-free survival (DFS). Tumors were classified as estrogen receptor (ER) -positive (n = 735, 59%) if immunohistochemical (IHC) or ligand-binding assays (LBA) were clearly positive. The ER-negative group included all other tumors (n = 511, 41%). A subset of the ER-negative group was defined as ER absent (n = 108, 9%) if IHC staining was none or if the LBA result was 0 fmol/mg cytosol protein. The median follow-up time was 7 years. RESULTS: Tamoxifen improved DFS in the ER-positive cohort (hazard ratio [HR] for tamoxifen v no tamoxifen = 0.59; 95% CI, 0.46 to 0.75; P < .0001) but not in the ER-negative cohort (HR = 1.02; 95% CI, 0.77 to 1.35; P = .89). Tamoxifen had a detrimental effect on patients with ER-absent tumors compared with no tamoxifen in an unplanned exploratory analysis (HR = 2.10; 95% CI, 1.03 to 4.29; P = .04). Patients with ER-positive tumors who achieved chemotherapy-induced amenorrhea had a significantly improved outcome (HR for amenorrhea v no amenorrhea = 0.61; 95% CI, 0.44 to 0.86; P = .004), whether or not they received tamoxifen. CONCLUSION: Tamoxifen after adjuvant chemotherapy significantly improved treatment outcome in premenopausal patients with endocrine-responsive disease, but its use as adjuvant therapy for patients with ER-negative tumors is not recommended.

Multicycle dose-intensive chemotherapy for women with high-risk primary breast cancer: results of International Breast Cancer Study Group Trial 15-95

PURPOSE: To compare adjuvant dose-intensive epirubicin and cyclophosphamide chemotherapy administered with filgrastim and progenitor cell support (DI-EC) with standard-dose anthracycline-based chemotherapy (SD-CT) for patients with early-stage breast cancer and a high risk of relapse, defined as stage II disease with 10 or more positive axillary nodes; or an estrogen receptor-negative or stage III tumor with five or more positive axillary nodes. PATIENTS AND METHODS: Three hundred forty-four patients were randomized after surgery to receive seven cycles of SD-CT over 22 weeks, or three cycles of DI-EC (epirubicin 200 mg/m2 plus cyclophosphamide 4 gm/m2 with filgrastim and progenitor cell support) over 6 weeks. All patients were assigned tamoxifen at the completion of chemotherapy. The primary end point was disease-free survival (DFS). RESULTS: After a median follow-up of 5.8 years (range, 3 to 8.4 years), 188 DFS events had occurred (DI-EC, 86 events; SD-CT, 102 events). The 5-year DFS was 52% for DI-EC and 43% for SD-CT, with hazard ratio of DI-EC compared with SD-CT of 0.77 (95% CI, 0.58 to 1.02; P = .07). The 5-year overall survival was 70% for DI-EC and 61% for SD-CT, with a hazard ratio of 0.79 (95% CI, 0.56 to 1.11; P = .17). There were eight cases (5%) of anthracycline-induced cardiomyopathy (two fatal) among those who received DI-EC. Women with hormone receptor-positive tumors benefited significantly from DI-EC. CONCLUSION: There was a trend in favor of DI-EC with respect to disease-free survival. A larger trial or meta-analysis will be required to reveal the true effect of dose-intensive therapy.

Identifying breast cancer patients at risk for Central Nervous System (CNS) metastases in trials of the International Breast Cancer Study Group (IBCSG)

BACKGROUND: We sought to determine whether a high-risk group could be defined among patients with operable breast cancer in whom a search of occult central nervous system (CNS) metastases was justified. PATIENTS AND METHODS: We evaluated data from 9524 women with early breast cancer (42% node-negative) who were randomized in International Breast Cancer Study Group clinical trials between 1978 and 1999, and treated without anthracyclines, taxanes, or trastuzumab. We identified patients whose site of first event was CNS and those who had a CNS event at any time. RESULTS: Median follow-up was 13 years. The 10-year incidence (10-yr) of CNS relapse was 5.2% (1.3% as first recurrence). Factors predictive of CNS as first recurrence included: node-positive disease (10-yr = 2.2% for > 3 N+), estrogen receptor-negative (2.3%), tumor size > 2 cm (1.7%), tumor grade 3 (2.0%), < 35 years old (2.2%), HER2-positive (2.7%), and estrogen receptor-negative and node-positive (2.6%). The risk of subsequent CNS recurrence was elevated in patients experiencing lung metastases (10-yr = 16.4%). CONCLUSION: Based on this large cohort we were able to define risk factors for CNS metastases, but could not define a group at sufficient risk to justify routine screening for occult CNS metastases.

ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Integrins and cardiovascular disease

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor alpha IIb beta 3, has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of alpha IIb beta 3 inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.

Treatment of premenopausal women with early breast cancer: old challenges and new opportunities

Breast cancer occurring in women before the age of menopause continues to be a major medical and psychological challenge. Endocrine therapy has emerged as the mainstay of adjuvant treatment for women with estrogen receptor-positive tumours. Although the suppression of ovarian function (by oophorectomy, irradiation of the ovaries or gonadotropin releasing factor analogues) is effective as adjuvant therapy if used alone, its value has not been proven after chemotherapy. This is presumably because of the frequent occurrence of chemotherapy-induced amenorrhoea. Tamoxifen reduces the risk of recurrence by approximately 40%, irrespective of age and the ovarian production of estrogens. The worth of ovarian function suppression in combination with tamoxifen is unproven and is being investigated in an intergroup randomised clinical trial (SOFT [Suppression of Ovarian Function Trial]). Aromatase inhibitors are more effective than tamoxifen in postmenopausal women but are only being investigated in younger patients. The use of chemotherapies is identical in younger and older patients; however, at present the efficacy of chemotherapy in addition to ovarian function suppression plus tamoxifen is unknown in premenopausal patients with endocrine responsive disease. 'Targeted' therapies such as monoclonal antibodies to human epidermal growth factor receptor (HER)-2, HER1 and vascular endothelial growth factor, 'small molecule' inhibitors of tyrosine kinases and breast cancer vaccines are rapidly emerging. Their use depends on the function of the targeted pathways and is presently limited to clinical trials. Premenopausal patients are best treated in the framework of a clinical trial.