Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR(+) tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth

An observational cohort study of the use of five-grass-pollen extract sublingual immunotherapy during the 2015 pollen season in France

Background:Allergic rhinitis affects around one quarter of the Western European population. Prophylactic allergen immunotherapy may be useful to reduce the risk of acute symptomatic attacks (hayfever). A five-grass pollen extract sublingual immunotherapy (5GPE-SLIT) has been developed for the treatment of allergic rhinitis to grass pollen. The objective of this study was to describe real-world treatment patterns with 5GPE-SLIT in France with respect to the prescribing information.Methods:This prospective cohort study was conducted by 90 community and hospital allergists. Adults and children (> 5 years old) starting a first treatment with 5GPE-SLIT prior to the 2015 pollen season were eligible. Data was collected at the inclusion visit and at the end of the pollen season. The primary outcome variable was compatibility of 5GPE-SLIT prescription with the prescribing information. This was determined with respect to four variables: (1) interval between 5GPE-SLIT initiation and onset of the pollen season ≥ 3 months, (2) age of patient ≥ 5 years, (3) intermittent symptoms or mild symptom severity (4) confirmatory diagnostic test. At study end, symptoms reported during the pollen season and any modifications to treatment or adverse events were documented.Results:280 adults and 203 children were enrolled. The prescribing information was respected for 82.5% of adults and 86.7% of children. A skin test was performed for all patients. 5GPE-SLIT was started 3-5 months before the pollen season for 85.3%. Treatment was discontinued before the start of the pollen season in 11.0% of patients overall, generally because of an adverse event (78.8% of discontinuations). The mean duration of treatment was 5.2 months in adults and 5.6 months in children. At the end of follow-up, symptoms during the pollen season were intermittent for 75.0% of adults and 85.7% of children, and severity was mild for 61.8 and 66.0% respectively. During 5GPE-SLIT, the following symptoms reported during the previous year were not reported again in > 50% of patients: nasal congestion, rhinorrhoea, repeated sneezing, conjunctivitis and nasal pruritus.Conclusions:5GPE-SLIT use was generally consistent with prescribing recommendations and was associated with an improvement of AR severity, with resolution of the principal AR symptoms in around half the patients treated.Trial registration EUPAS9358. Registered 13 May 2015. Not prospectively registered. http://www.encepp.eu/encepp/viewResource.htm?id=16229

Genome-wide analysis of 53,400 people with irritable bowel syndrome highlights shared genetic pathways with mood and anxiety disorders

Funder: Kennedy Trust Rheumatology Research Prize StudentshipFunder: DFG Cluster of Excellence “Precision Medicine in Chronic In-flammation” (PMI; ID: EXC2167)Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: “Ideas” Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): 715772Funder: NWO-VIDI grant 016.178.056, the Netherlands Heart Foundation CVON grant 2018-27, and NWO Gravitation grant ExposomeNLFunder: Li Ka Shing Foundation (Li Ka Shing Foundation Limited); doi: https://doi.org/10.13039/100007421Abstract: Irritable bowel syndrome (IBS) results from disordered brain–gut interactions. Identifying susceptibility genes could highlight the underlying pathophysiological mechanisms. We designed a digestive health questionnaire for UK Biobank and combined identified cases with IBS with independent cohorts. We conducted a genome-wide association study with 53,400 cases and 433,201 controls and replicated significant associations in a 23andMe panel (205,252 cases and 1,384,055 controls). Our study identified and confirmed six genetic susceptibility loci for IBS. Implicated genes included NCAM1, CADM2, PHF2/FAM120A, DOCK9, CKAP2/TPTE2P3 and BAG6. The first four are associated with mood and anxiety disorders, expressed in the nervous system, or both. Mirroring this, we also found strong genome-wide correlation between the risk of IBS and anxiety, neuroticism and depression (rg > 0.5). Additional analyses suggested this arises due to shared pathogenic pathways rather than, for example, anxiety causing abdominal symptoms. Implicated mechanisms require further exploration to help understand the altered brain–gut interactions underlying IBS

DKAT cell line karyotype.

<p>A representative mitotic DKAT cell from sideline 1 is shown. <b>A.</b> Metaphase cell with chromosomes shown in SKY display colors. <b>B.</b> Inverted and contrast-enhanced DAPI image of the same metaphase cell. <b>C.</b> The same metaphase cell with chromosomes shown in spectra-based classification colors. <b>D.</b> Spectral karyotype of the same metaphase cell shown in SKY display colors.</p

Differential gene expression (MEGM/SCGM).

<p>List of genes related to the epithelial phenotype, cell-cell junctions, and motility with altered expression following 14 days of culture in SCGM to induce EMT. Microarray experiments were performed as described in Materials and Methods.</p

DKAT tumorsphere culture.

<p>Immunofluorescence of DKAT tumorspheres stained for E-cadherin, β-catenin, CK17 and CK18 (upper and middle panels). Tumorspheres were also dual stained for E-cadherin (red) and vimentin (green), or CK5 (red) and CK18 (green) (lower panel). Images were acquired with a 63 × objective. Scale bar = 40 µm.</p

Basal/myoepithelial differentiation of DKAT xenografts. A.

<p>DKAT xenograft tumor stained with DAPI (blue) nuclear counter stain, CK14 (green) showing basal/myoepithelial differentiation of tumor cells, and human-specific p53 (red) confirming that both the CK14-positive cells and the more luminal, CK14 weak/negative cells are human DKAT tumor cells. 100 micron scale bar in the DAPI channel applies to all four images. <b>B.</b> DKAT tumor from a second mouse at higher magnification shows an area of vague glandular architecture with somewhat elongated CK14-positive basal/myoepithelial cells (arrows) surrounding CK14 weak/negative cells which surround a poorly formed glandular lumen (asterix). 25 micron scale bar show in in DAPI channel.</p

Characterization of human primary tumor, chest wall recurrence and bone marrow metastasis.

<p>Qualitative evaluation based on <i>in situ</i> hybridization for Her2, and based on immunohistochemistry for all other markers.</p

Human breast cancer specimen displays morphological and biochemical evidence suggestive of EMT and MET.

<p>Images of the patient’s primary tumor focus #3 (top row), focus #6 (second row), chest wall recurrence (third row), and bone marrow biopsy (bottom row) showing H&E staining and immunohistochemistry for CK5, CK8/18, vimentin, and E-cadherin.</p

Evidence for morphologic and phenotypic changes consistent with <i>in vitro</i> EMT.

<p><b>A.</b> Phase contrast or immunofluorescence images of DKAT cells cultured in either MEGM (upper) or SCGM for 14 days (lower). Phase contrast images were acquired with a 20 × objective, scale bar = 40 µm. IF images were acquired with a 40 × objective, scale bar = 20 µm. Cells were stained simultaneously for vimentin (green) and E-cadherin (red), or CK18 (green) and CK5 (red). <b>B.</b> E-cadherin immunofluorescence images of DKAT cells cultured in MEGM or SCGM for 2 hours. <b>C.</b> Total cell lysates from DKAT cells cultured in MEGM or SCGM for 1 h, 24 h, or 14 days were analyzed by western blot for vimentin, E-cadherin and actin. <b>D.</b> Scratch wound healing assay comparing DKAT cells grown in MEGM or SCGM (14 days) and MDA-MB-231 cells. Images (upper panel) were taken at the time of the scratch (T = 0) and 12 hours later with a 5 × objective. Scale bar = 200 µm. Results (lower panel) are expressed as the percentage of the scratch area closed at 12 hrs as measured by ImageJ software, and are an average of 12 scratches per sample. <b>E.</b> Total cell lysates from a clonal DKAT cell line cultured in MEGM or SCGM (14 days) were analyzed by western blot for vimentin, E-cadherin and actin. <b>F.</b> Total cell lysate from passage matched cultures of DKAT cells grown in MEGM, SCGM, or SCGM for 10 passages and then returned to MEGM for 5 passages were analyzed by western blot for epithelial and mesenchymal markers. <b>G.</b> Immunofluorescence (IF) images of subcloned DKAT-SCGM cells cultured in SCGM or MEGM for 14 days. Cells were stained simultaneously for vimentin (green) and E-cadherin (red), and images were acquired with a 40 × objective. <b>H.</b> Total cell lysates from the indicated cell lines cultured in their normal media or switched to MEGM or SCGM for 14 days were analyzed by western blot for vimentin, E-cadherin, and actin expression. N = Normal, M = MEGM, S = SCGM.</p