Gallbladder Cancer Risk and Indigenous South American Mapuche Ancestry: Instrumental Variable Analysis Using Ancestry-Informative Markers.

A strong association between the proportion of indigenous South American Mapuche ancestry and the risk of gallbladder cancer (GBC) has been reported in observational studies. Chileans show the highest incidence of GBC worldwide, and the Mapuche are the largest indigenous people in Chile. We set out to assess the confounding-free effect of the individual proportion of Mapuche ancestry on GBC risk and to investigate the mediating effects of gallstone disease and body mass index (BMI) on this association. Genetic markers of Mapuche ancestry were selected based on the informativeness for assignment measure, and then used as instrumental variables in two-sample Mendelian randomization analyses and complementary sensitivity analyses. Results suggested a putatively causal effect of Mapuche ancestry on GBC risk (inverse variance-weighted (IVW) risk increase of 0.8% per 1% increase in Mapuche ancestry proportion, 95% CI 0.4% to 1.2%, p = 6.7 × 10-5) and also on gallstone disease (3.6% IVW risk increase, 95% CI 3.1% to 4.0%), pointing to a mediating effect of gallstones on the association between Mapuche ancestry and GBC. In contrast, the proportion of Mapuche ancestry showed a negative effect on BMI (IVW estimate -0.006 kg/m2, 95% CI -0.009 to -0.003). The results presented here may have significant implications for GBC prevention and are important for future admixture mapping studies. Given that the association between the individual proportion of Mapuche ancestry and GBC risk previously noted in observational studies appears to be free of confounding, primary and secondary prevention strategies that consider genetic ancestry could be particularly efficient.

Development and internal validation of a multifactorial risk prediction model for gallbladder cancer in a high-incidence country.

Since 2006, Chile has been implementing a gallbladder cancer (GBC) prevention program based on prophylactic cholecystectomy for gallstone patients aged 35 to 49 years. The effectiveness of this prevention program has not yet been comprehensively evaluated. We conducted a retrospective study of 473 Chilean GBC patients and 2137 population-based controls to develop and internally validate three GBC risk prediction models. The Baseline Model accounted for gallstones while adjusting for sex and birth year. Enhanced Model I also included the non-genetic risk factors: body mass index, educational level, Mapuche surnames, number of children and family history of GBC. Enhanced Model II further included Mapuche ancestry and the genotype for rs17209837. Multiple Cox regression was applied to assess the predictive performance, quantified by the area under the precision-recall curve (AUC-PRC) and the number of cholecystectomies needed (NCN) to prevent one case of GBC at age 70 years. The AUC-PRC for the Baseline Model (0.44%, 95%CI 0.42-0.46) increased by 0.22 (95%CI 0.15-0.29) when non-genetic factors were included, and by 0.25 (95%CI 0.20-0.30) when incorporating non-genetic and genetic factors. The overall NCN for Chileans with gallstones (115, 95%CI 104-131) decreased to 92 (95%CI 60-128) for Chileans with a higher risk than the median according to Enhanced Model I, and to 80 (95%CI 59-110) according to Enhanced Model II. In conclusion, age, sex and gallstones are strong risk factors for GBC, but consideration of other non-genetic factors and individual genotype data improves risk prediction and may optimize allocation of financial resources and surgical capacity.

Identification of Circulating lncRNAs Associated with Gallbladder Cancer Risk by Tissue-Based Preselection, Cis-eQTL Validation, and Analysis of Association with Genotype-Based Expression.

Long noncoding RNAs (lncRNAs) play key roles in cell processes and are good candidates for cancer risk prediction. Few studies have investigated the association between individual genotypes and lncRNA expression. Here we integrate three separate datasets with information on lncRNA expression only, both lncRNA expression and genotype, and genotype information only to identify circulating lncRNAs associated with the risk of gallbladder cancer (GBC) using robust linear and logistic regression techniques. In the first dataset, we preselect lncRNAs based on expression changes along the sequence "gallstones → dysplasia → GBC". In the second dataset, we validate associations between genetic variants and serum expression levels of the preselected lncRNAs (cis-lncRNA-eQTLs) and build lncRNA expression prediction models. In the third dataset, we predict serum lncRNA expression based on individual genotypes and assess the association between genotype-based expression and GBC risk. AC084082.3 and LINC00662 showed increasing expression levels (p-value = 0.009), while C22orf34 expression decreased in the sequence from gallstones to GBC (p-value = 0.04). We identified and validated two cis-LINC00662-eQTLs (r2 = 0.26) and three cis-C22orf34-eQTLs (r2 = 0.24). Only LINC00662 showed a genotyped-based serum expression associated with GBC risk (OR = 1.25 per log2 expression unit, 95% CI 1.04-1.52, p-value = 0.02). Our results suggest that preselection of lncRNAs based on tissue samples and exploitation of cis-lncRNA-eQTLs may facilitate the identification of circulating noncoding RNAs linked to cancer risk.

NGF/TRKA Promotes ADAM17-Dependent Cleavage of P75 in Ovarian Cells: Elucidating a Pro-Tumoral Mechanism.

Nerve growth factor (NGF) and its high-affinity receptor TRKA are overexpressed in epithelial ovarian cancer (EOC) displaying a crucial role in the disease progression. Otherwise, NGF interacts with its low-affinity receptor P75, activating pro-apoptotic pathways. In neurons, P75 could be cleaved by metalloproteinases (α and γ-secretases), leading to a decrease in P75 signaling. Therefore, this study aimed to evaluate whether the shedding of P75 occurs in EOC cells and whether NGF/TRKA could promote the cleavage of the P75 receptor. The immunodetection of the α-secretase, ADAM17, TRKA, P75, and P75 fragments was assessed by immunohisto/cytochemistry and Western blot in biopsies and ovarian cell lines. The TRKA and secretases' inhibition was performed using specific inhibitors. The results show that P75 immunodetection decreased during EOC progression and was negatively correlated with the presence of TRKA in EOC biopsies. NGF/TRKA increases ADAM17 levels and the fragments of P75 in ovarian cells. This effect is abolished when cells are previously treated with ADAM17, γ-secretase, and TRKA inhibitors. These results indicate that NGF/TRKA promotes the shedding of P75, involving the activation of secretases such as ADAM17. Since ADAM17 has been proposed as a screening marker for early detection of EOC, our results contribute to understanding better the role of ADAM17 and NGF/TRKA in EOC pathogenesis, which includes the NGF/TRKA-mediated cleavage of P75.

Fatores associados ao uso clínico da Classificação Internacional de Funcionalidade, Incapacidade e Saúde por fisioterapeutas: estudo survey exploratório / Factors associated with the clinical use of the International Classification of Functioning, Disability and Health by physical therapists: an exploratory survey

Objetivo: Identificar os fatores contextuais (i.e., fatores pessoais e fatores relacionados ao trabalho) associados ao uso clínico da Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIF) por fisioterapeutas. Métodos: Foi realizado um estudo exploratório do tipo survey. O questionário Barreiras pessoais e ambientais para implementação da CIF por fisioterapeutas" foi enviado aos fisioterapeutas do Espírito Santo, Brasil. Análise de regressão logística foi usada para explorar associações entre características pessoais e organizacionais com educação, atitudes, crenças, interesses, percepção de suporte e disponibilidade de recursos no ambiente de trabalho para uso da CIF na prática clínica. Resultados: A taxa de resposta para a maioria das análises foi 46% (n= 59). Seis características explicaram 35% da variação associada a interesses, atitudes e crenças para o uso da CIF (X²= 16.6, p= 0.01). Três características explicaram 24% da variação associada à educação para o uso da CIF (X²= 8.9, p= 0.03). Três características explicaram 61% da variação associada à percepção de suporte e disponibilidade de recursos para o uso da CIF (X²= 30.5, p<0.01). A barreira mais citada foi dificuldade ao aplicar a CIF para avaliar pacientes (62%). Conclusão: Os fisioterapeutas possuem atitudes positivas em relação ao uso da CIF, mas a grande maioria não recebeu formação acadêmica adequada e reporta auto-eficácia insuficiente para seu uso na prática clínica. É necessária a adoção de uma infraestrutura organizacional que endosse o uso clínico da CIF alinhada à modificação nos currículos de formação e capacitação continuada.

Objective: To identify the contextual factors (i.e., personal, and work-related factors) associated with the clinical use of the International Classification of Functioning, Disability and Health (ICF) by physical therapists. Method: A cross-sectional exploratory survey was carried out. The questionnaire Personal and environmental barriers for the implementation of the ICF by physical therapists was mailed to physical therapists living in Espírito Santo, Brazil. Logistic regression analysis was used to explore associations between personal and work-related characteristics with education, attitudes, beliefs, interest, and perception related to work resources for applying the ICF. Results: The response rate for most analyses was 46% (n= 59). Six characteristics explained 35% of the variance associated with interests, attitudes, and beliefs for the use of the ICF (X²= 16.6, p= 0.01). Three characteristics explained 24% of the variance associated with education for the use of ICF (X²= 8.9, p= 0.03). Three characteristics explained 61% of the variance associated with the perception of support and availability of resources for the use of the ICF (X²= 30.5, p<0.01). The most cited barrier was difficulty in applying the ICF for assessments of patients (62%). Conclusions: Physical therapists have positive attitudes towards the use of ICF, but most respondents did not receive satisfactory academic training and reported insufficient self-efficacy for using ICF in clinical practice. The adoption of an organizational infrastructure that endorses the clinical use of the ICF in association with an updated and continued education is required.

Epigenome-Wide Analysis of Methylation Changes in the Sequence of Gallstone Disease, Dysplasia, and Gallbladder Cancer.

BACKGROUND AND AIMS: Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low-income and middle-income countries, and research into this disease has long been limited. In this study we therefore investigate the epigenetic changes along the model of GBC carcinogenesis represented by the sequence gallstone disease → dysplasia → GBC in Chile, the country with the highest incidence of GBC worldwide. APPROACH AND RESULTS: To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of formalin-fixed, paraffin-embedded gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Preprocessed, quality-controlled data from 82 samples (gallstones n = 32, low-grade dysplasia n = 13, high-grade dysplasia n = 9, GBC n = 28) were available to identify differentially methylated markers, regions, and pathways as well as changes in copy number variations (CNVs). The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of cytosine-guanine dinucleotide islands and gene promoter regions. The methylation of genes implicated in Wnt signaling, Hedgehog signaling, and tumor suppression increased with tumor grade. CNVs also increased with GBC development and affected cyclin-dependent kinase inhibitor 2A, MDM2 proto-oncogene, tumor protein P53, and cyclin D1 genes. Gains in the targetable Erb-B2 receptor tyrosine kinase 2 gene were detected in 14% of GBC samples. CONCLUSIONS: Our results indicate that GBC carcinogenesis comprises three main methylation stages: early (gallstone disease and low-grade dysplasia), intermediate (high-grade dysplasia), and late (GBC). The identified gradual changes in methylation and CNVs may help to enhance our understanding of the mechanisms underlying this aggressive disease and eventually lead to improved treatment and early diagnosis of GBC.

Gallstones, Body Mass Index, C-Reactive Protein, and Gallbladder Cancer: Mendelian Randomization Analysis of Chilean and European Genotype Data.

BACKGROUND AND AIMS: Gallbladder cancer (GBC) is a neglected disease with substantial geographical variability: Chile shows the highest incidence worldwide, while GBC is relatively rare in Europe. Here, we investigate the causal effects of risk factors considered in current GBC prevention programs as well as C-reactive protein (CRP) level as a marker of chronic inflammation. APPROACH AND RESULTS: We applied two-sample Mendelian randomization (MR) using publicly available data and our own data from a retrospective Chilean and a prospective European study. Causality was assessed by inverse variance weighted (IVW), MR-Egger regression, and weighted median estimates complemented with sensitivity analyses on potential heterogeneity and pleiotropy, two-step MR, and mediation analysis. We found evidence for a causal effect of gallstone disease on GBC risk in Chileans (P = 9 × 10-5 ) and Europeans (P = 9 × 10-5 ). A genetically elevated body mass index (BMI) increased GBC risk in Chileans (P = 0.03), while higher CRP concentrations increased GBC risk in Europeans (P = 4.1 × 10-6 ). European results suggest causal effects of BMI on gallstone disease (P = 0.008); public Chilean data were not, however, available to enable assessment of the mediation effects among causal GBC risk factors. CONCLUSIONS: Two risk factors considered in the current Chilean program for GBC prevention are causally linked to GBC risk: gallstones and BMI. For Europeans, BMI showed a causal effect on gallstone risk, which was itself causally linked to GBC risk.

ABCB1/4 gallbladder cancer risk variants identified in India also show strong effects in Chileans.

BACKGROUND: The first large-scale genome-wide association study of gallbladder cancer (GBC) recently identified and validated three susceptibility variants in the ABCB1 and ABCB4 genes for individuals of Indian descent. We investigated whether these variants were also associated with GBC risk in Chileans, who show the highest incidence of GBC worldwide, and in Europeans with a low GBC incidence. METHODS: This population-based study analysed genotype data from retrospective Chilean case-control (255 cases, 2042 controls) and prospective European cohort (108 cases, 181 controls) samples consistently with the original publication. RESULTS: Our results confirmed the reported associations for Chileans with similar risk effects. Particularly strong associations (per-allele odds ratios close to 2) were observed for Chileans with high Native American (=Mapuche) ancestry. No associations were noticed for Europeans, but the statistical power was low. CONCLUSION: Taking full advantage of genetic and ethnic differences in GBC risk may improve the efficiency of current prevention programs.

The nerve growth factor alters calreticulin translocation from the endoplasmic reticulum to the cell surface and its signaling pathway in epithelial ovarian cancer cells.

Ovarian cancer is the seventh most common cancer among women worldwide, causing approximately 120,000 deaths every year. Immunotherapy, designed to boost the body's natural defenses against cancer, appears to be a promising option against ovarian cancer. Calreticulin (CRT) is an endoplasmic reticulum (ER) resident chaperone that, translocated to the cell membrane after ER stress, allows cancer cells to be recognized by the immune system. The nerve growth factor (NGF) is a pro-angiogenic molecule overexpressed in this cancer. In the present study, we aimed to determine weather NGF has an effect in CRT translocation induced by cytotoxic and ER stress. We treated A2780 ovarian cancer cells with NGF, thapsigargin (Tg), an ER stress inducer and mitoxantrone (Mtx), a chemotherapeutic drug; CRT subcellular localization was analyzed by immunofluorescence followed by confocal microscopy. In order to determine NGF effect on Mtx and Tg-induced CRT translocation from the ER to the cell membrane, cells were preincubated with NGF prior to Mtx or Tg treatment and CRT translocation to the cell surface was determined by flow cytometry. In addition, by western blot analyses, we evaluated proteins associated with the CRT translocation pathway, both in A2780 cells and human ovarian samples. We also measured NGF effect on cell apoptosis induced by Mtx. Our results indicate that Mtx and Tg, but not NGF, induce CRT translocation to the cell membrane. NGF, however, inhibited CRT translocation induced by Mtx, while it had no effect on Tg-induced CRT exposure. NGF also diminished cell death induced by Mtx. NGF effect on CRT translocation could have consequences in immunotherapy, potentially lessening the effectiveness of this type of treatment.

Effect of estradiol on the expression of angiogenic factors in epithelial ovarian cancer.

INTRODUCTION: Ovarian cancer presents a high angiogenesis (formation of new blood vessels) regulated by pro-angiogenic factors, mainly vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). An association between endogenous levels of estrogen and increased risk of developing ovarian cancer has been reported. Estrogen action is mediated by the binding to its specific receptors (ERα and ERß), altered ERα/ERß ratio may constitute a marker of ovarian carcinogenesis progression. OBJECTIVE: To determine the effect of estradiol through ERα on the expression of NGF and VEGF in epithelial ovarian cancer (EOC). METHODOLOGY: Levels of phosphorylated estrogen receptor alpha (pERα) were evaluated in well, moderate and poorly differentiated EOC samples (EOC-I, EOC-II, EOC-III). Additionally, ovarian cancer explants were stimulated with NGF (0, 10 and 100 ng/ml) and ERα, ERß and pERα levels were detected. Finally, human ovarian surface epithelial (HOSE) and epithelial ovarian cancer (A2780) cell lines were stimulated with estradiol, where NGF and VEGF protein levels were evaluated. RESULTS: In tissues, ERs were detected being pERα levels significantly increased in EOC-III samples compared with EOC-I (p<0.05). Additionally, ovarian explants treated with NGF increased pERα levels meanwhile total ERα and ERß levels did not change. Cell lines stimulated with estradiol revealed an increase of NGF and VEGF protein levels (p<0.05). CONCLUSIONS: Estradiol has a positive effect on pro-angiogenic factors such as NGF and VEGF expression in EOC, probably through the activation of ERα; generating a positive loop induced by NGF increasing pERα levels in epithelial ovarian cells.

Altered Steroid Metabolism and Insulin Signaling in PCOS Endometria: Impact in Tissue Function.

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine/ metabolic disorder characterized by hyperandrogenemia and in most cases, by hyperinsulinemia in addition to obesity. Besides ovarian dysfunction, endometrial physiology is also disrupted since this tissue is highly dependent on the action of steroids; in case of conception cycles, high percentage of abortion is observed. Because of the endocrine/metabolic alterations, PCOS-women present high probability to develop hyperplasia and endometrial cancer, where an imbalance of cell proliferation/apoptosis processes is detected. Additionally, insulin pathway and the endometrial energetic homeostasis are also compromised. METHODS: The aim of this review was to report molecular alterations related to insulinresistance and/or obesity in PCOS-women endometria that could drive to infertility. For this, several methods were employed: immunohistocytochemistry, qPCR, western-blot, glucoseuptake, cell cultures, among others. RESULTS: Diminished levels and activity of several insulin signaling pathway molecules, like IRS-1/AS160/PKCζ, were detected. Concomitantly, a defect in the synthesis and GLUT4 translocation to cell surface is induced. Oral administration of metformin (insulin sensitizer) to PCOS-patients increases GLUT4 endometrial levels, improving fertility of those patients. Another relevant feature is the high percentage of obesity in PCOS-women; adiponectin is an obesity marker and elicits an insulin-sensitizer action, being diminished in plasma of obese PCOSwomen similar to its endometrial level, adiponectin-receptors and APPL1, an adapter molecule of adiponectin pathway. Moreover, obesity and PCOS can induce a pro-inflammatory environment, exaggerating the alterations in insulin pathway. CONCLUSION: The evidences obtained in PCOS-endometria clearly indicate that these molecular defects could partially explain the reproductive failures of these patients.

Hyperandrogenism Decreases GRP78 Protein Level and Glucose Uptake in Human Endometrial Stromal Cells.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) exhibit a low fertility by chronic hyperandrogenemia. Different evidence have shown that androgens could regulate the endoplasmic reticulum (ER) homeostasis and glucose metabolism. However, it is unclear whether androgens can exert these effects on human endometrial stromal cells. Our goal was to study the protein content of GRP78 (an ER homeostasis marker) in endometria from women with PCOS and healthy women and to assess the GRP78 protein levels and its relationship with glucose uptake on a human endometrial stromal cell line stimulated with testosterone. METHODS: Immunohistochemistry assays for GRP78 were performed on endometrial samples obtained from women with PCOS (n = 8) and control women subjected to hysterectomy (n = 8). Western blot analysis for GRP78 and glucose uptake was assessed in a telomerase-immortalized human endometrial stromal cell line (T-HESC) exposed to testosterone for 24 or 48 hours and challenged to an insulin short-term stimulation. Tukey test was performed for human samples comparison. Student t test or ANOVA-Bonferroni test was carried out according to the in vitro experiment. P < .05 was considered as significant. RESULTS: GRP78 stromal immunostaining was reduced in PCOS endometria compared to controls (P < .05). The T-HESC shows a testosterone-dependent downregulation of GRP78 protein content (P < .05), concomitant with half-reduction in glucose uptake compared to controls (P < .05). Moreover, enhanced small interfering RNA against GRP78 messenger RNA leads to a decrease in glucose uptake (P < .05). Such effects were reverted by hydroxyflutamide, an inhibitor of androgen receptor. CONCLUSION: These results suggest that hyperandrogenemic PCOS environment could compromise the endometrial homeostasis confirmed by the decrease in glucose uptake induced by testosterone and exhibited by stromal cells.

Role of dihydrotestosterone (DHT) on TGF-ß1 signaling pathway in epithelial ovarian cancer cells.

PURPOSE: One of the hypotheses regarding the genesis of epithelial ovarian cancer involves the action of androgens on the proliferation of epithelial ovarian cells, as well as inclusion cysts. The purpose of the present study was to evaluate whether DHT causes changes in the TGF-ß1 pathway that might modify the anti-proliferative effect of the latter. METHODS: The levels of TGF-ß1 protein, of its receptors (TGFBR1 and TGFBR2), of Smad2/3 (canonical signaling pathway protein) and of p21 (cell cycle protein) were assessed in ovarian tissues, epithelial ovarian cancer cell lines (A2780) and control cell lines (HOSE) through the use of immunohistochemistry and immunocytochemistry. Additionally, cell lines were treated with 100 nmol/L DHT, 10 ng/mL of TGF-ß1 and DHT + TGF-ß1 during 72 h in the presence and absence of a siRNA against androgen receptor. After treatment, TGFBR1 and TGFBR2 levels were detected through Western blotting and p21 was assessed through immunocytochemistry. RESULTS: Epithelial ovarian cancer tissues showed a decrease in TGF-ß1 I receptor (p < 0.05) and a change in Smad2/3 protein levels. Additionally, after treatment of cell lines with DHT, protein levels of TGF-ß1 receptors (TGFBR1-TGFBR2) showed a decrease (p < 0.05) that might cause a potential disorder in TGF-ß1 response, represented by the significant decrease in p21 protein levels in the presence of DHT (p < 0.001). CONCLUSIONS: Overall, our results indicate a defect in the canonical TGF-ß signaling pathway in epithelial ovarian cancer caused by androgen action, thus suggesting eventual changes in such tissue proliferation rates.

Expression of steroid sulfated transporters and 3ß-HSD activity in endometrium of women having polycystic ovary syndrome.

Intracrinology mechanism involves the metabolism of steroids in peripheral tissues, such as DHEA, to molecules with estrogenic or androgenic activity. Proliferation rate of endometria from Polycystic Ovary Syndrome women (PCOS) is increased, favoring hyperplasia development. Besides, in endometria from PCOS-women the synthesis of androst-5-ene-3ß,17ß-diol (androstenediol), an estrogenic molecule, is enhanced concomitantly to increased cellular proliferation. DHEA, the major intracrinological precursor, circulates mainly in its sulfated form and requires transporters for cell intake, that belong to the families of organic anion transporting polypeptides (OATP) and organic anion transporters (OAT). The aim of this study was to determine protein levels and activity of sulfated steroid transporters OATP2B1, OATP3A1, OATP4A1 and OAT4 in endometria from control and PCOS-women and to evaluate the activity of the enzyme 3ß-HSD. Levels of transporters were done by RT-PCR (OAT4 only) and Western-blot (WB). Additionally, in primary culture cells stimulated with steroids, protein levels by WB and uptake of tritiated DHEAS, were evaluated; 3ß-HSD activity was assessed using radiolabel substrate. PCOS-endometrium had higher levels of OATP2B1 and OATP4A1 than CE (p<0.05); decreased OATP4A1 levels were found in androstenediol or testosterone-stimulated cells. Accordingly, the entry of DHEAS to cells was lower in cells stimulated with testosterone (p<0.05); 3ß-HSD-activity was similar in control and PCOS-endometria. Therefore, this study describes that steroids can modulate the expression and activity of transporters of OATPs-family in human endometria and that some transporter levels are increased in PCOS-endometria, suggesting a potential role in the pathogenesis of endometrial hyperplasia of these patients.

Enhanced caveolin-1 expression increases migration, anchorage-independent growth and invasion of endometrial adenocarcinoma cells.

BACKGROUND: Caveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease. METHODS: Here, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4ß-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar. RESULTS: Immunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4ß-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4ß-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells. CONCLUSION: Our study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth.

Tumores estromales de potencial maligno incierto (STUMP): descripción epidemiológica y manejo durante el período 2004-2013 / Stromal tumors of an uncertain malignant potential (STUMP): epidemiological description and handling during the 2004-2013 period

Los tumores estromales de potencial maligno incierto (STUMP) se enmarcan dentro de las lesiones de células fusadas de la próstata, entidades de relativa infrecuencia en la práctica urológica habitual, contabilizándose entre 0,1-0,2 por ciento 1 de todas los tumores malignos prostáticos. Corresponden a una proliferación no epitelial mesenquimática de células fusadas estromales, que pueden adquirir la capacidad de infiltrar el epitelio glandular prostático. Representan un desafío tanto para el patólogo, por la dificultad diagnóstica, como para el clínico, pues no existe consenso respecto a su manejo. Materiales y métodos: estudio retrospectivo descriptivo. Se filtró la base de datos del servicio de Anatomía Patología del Hospital San Borja Arriarán con los términos clave “STUMP”, “Tumor estromal próstata”, tanto de biopsias transrrectales como de piezas operatorias. De los pacientes con diagnóstico histológico e inmunohistoquímico compatible, se registraron características epidemiológicas, estudio, manejo y sobrevida global. Resultados: se encontraron 3(tres) pacientes con diagnóstico histológico de STUMP. Edad promedio al diagnóstico 66años. Dos de los casos (66,6 ´po0r ciento) correspondieron a hallazgo en biopsia diferida por cirugía benigna prostática, (una por Adenomectomía transvesical y otra por RTU-P). El tercer caso correspondió a un hallazgo en biopsia por punción transrrectal ecodirigida, en contexto de PSA elevado (100ng/mL) y 4 biopsias previas: 3 normales, y una con informe de posible sarcoma prostático. La terapia de elección en 2 casos fue seguimiento y en uno se indicó cirugía radical. En los casos descritos no se registra mortalidad, con seguimiento promedio de 32 meses. Conclusión: Los tumores estromales de potencial maligno incierto son un diagnóstico histológico infrecuente, descrito como hallazgo tanto en la biopsia transrrectal como de pieza quirúrgica. No existen consensos internacionales ni guías de manejo. En nuestra experiencia...

Stromal tumors of uncertain malignant potential (STUMP) are included in the spindle cell lesions of the adult prostate, a very rare diagnostic entity in the clinical practice, accounting no more than 0,1-0,2 percent of all prostate malignancies. STUMP is defined as a non epithelial proliferation of spindle cells, which can acquire the ability of infiltrate the prostate glands. This lesions represent a challenge for both clinical and pathology physician. Material and methods: retrospective cohort study. A research was performed at the San Borja Hospital Pathology Service’s database, using keywords “STUMP”, “Stromal prostate tumor”. From eligible patients we took epidemiology and clinical data. Results: a total of three patients were included. Average age was 66 years. STUMP was founded in two patients in the pathology report after benign prostatic surgery (Simple prostatectomy and TUR-P). A third patient was diagnosed by a needle biopsy (TRUS Biopsy). This patient had an elevated PSA (>100 ng/mL), 4 previous biopsies, three normal and a fourth suspicious of prostate sarcoma. Surveillance was the therapy chosen for those patients diagnosed with STUMP after benign surgery. Radical prostatectomy was offered to the third patient. Average follow up was 32 month Conclusion: STUMP is a rare diagnostic, and is a challenge for both clinical and pathology physician. There are no guidelines for the management. In our series we confirmed the low frequency of this condition, and the management in our service is similar as other series described in literature...

The role of androst-5-ene-3ß,17ß-diol (androstenediol) in cell proliferation in endometrium of women with polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) show high prevalence of endometrial hyperplasia and adenocarcinoma. Endometrial proliferation is increased, evaluated by high levels of Ki67 (cell cycle marker) and low levels of p27 (negative regulator of cell cycle). Nevertheless, endometrial changes in cyclin D1 (positive regulator of cell cycle) in PCOS-women are not described. Androst-5-ene-3ß,17ß-diol (androstenediol), steroid with estrogenic activity present in endometria, could be related to increased endometrial cell proliferation. The objective of this study was to determine protein content of cyclin D1 and androstenediol levels in endometria from PCOS and control-women and to evaluate the possible mechanism favoring cell proliferation associated with hormonal characteristics of patients. Therefore, cyclin D1 protein content in PCOS-women and control-endometrial tissue were assessed by western blot and immunohistochemistry. The androstenediol levels were evaluated by ELISA. To further analyze the effect of steroids (androstenediol, 17ß-estradiol, testosterone) in cell proliferation, levels of proteins cyclin D1, p27 and Ki67 were evaluated in an in vitro model of stromal endometrial cells T-HESC and St-T1b. An increase in cyclin D1 and androstenediol was observed in tissues from PCOS-women relative to control group (p<0.05). In the in vitro model, androstenediol exerted increase in cyclin D1 (p<0.05) and a decrease in p27 protein level (p<0.05), while Ki67 in St-T1b cells increased under this stimulus (p<0.05). Testosterone produces opposite effects in the levels of the above markers (p<0.05). Therefore, the hormonal imbalance associated with this syndrome could alter endometrial tissue homeostasis, promoting cell proliferation. Androstenediol is a molecule that could be involved by stimulating proliferation, whereas testosterone elicits a role of cell cycle repressor.

Metformin augments the levels of molecules that regulate the expression of the insulin-dependent glucose transporter GLUT4 in the endometria of hyperinsulinemic PCOS patients.

STUDY QUESTION: Does treatment with the insulin sensitizer metformin modify the levels and activation of proteins related to the expression of the insulin-dependent glucose transporter (GLUT4), such as adenosine monophosphate-activated protein kinase (AMPK) and myocyte enhancer factor 2A (MEF2A), in endometria from hyperinsulinemic hyperandrogenemic polycystic ovary syndrome (PCOS h-Ins) patients? SUMMARY ANSWER: In PCOS h-Ins patients, metformin increases endometrial levels of GLUT4 mRNA and protein levels by normalizing the quantity and activation of molecules that regulate GLUT4 expression to healthy values. These changes could improve endometrial metabolic function. WHAT IS ALREADY KNOWN: PCOS is an endocrine-metabolic disorders closely associated with insulin resistance. In particular, the insulin signaling pathway is impaired in endometria from these patients and the concentration of GLUT4, as well as the molecules involved in its translocation to the cell surface, is decreased. However, there are limited data about the mechanisms that regulate the GLUT4 expression in the endometria and the effect of metformin on them. STUDY DESIGN, SIZE AND DURATION: This is a case-control study in the setting of a research unit, approved by the Ethical Committees of our institution. The groups whose endometria were studied were PCOS h-Ins (n = 8); PCOS patients with hyperandrogenemia hyperinsulinemia taking only metformin for at least 3 months (PCOS-MTF, n = 8) and healthy fertile women at the time of hysterectomy because of benign pathology as controls (CE, n = 8). PARTICIPANTS/MATERIALS, SETTING, METHODS: Steroids and sex hormone-binding globulin were measured and glucose and insulin levels were evaluated during an oral glucose tolerance test. Protein levels for αAMPK (catalytic subunit of AMPK), phosphorylated (p)-AMPKαThr(172) (activating phosphorylation site), MEF2A, p-MEF2AThr312 (activating phosphorylation site) and GLUT4 were assessed by western blot and immunohistochemistry. In addition, GLUT4 gene expression was evaluated by RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: We found significantly lower levels of MEF2A and p-MEF2AThr312 in PCOS h-Ins compared with CE endometria (P < 0.05). Also, we detected lower levels of p-AMPKαThr(172) in PCOS h-Ins endometria compared with the PCOS-MTF group (P < 0.05). The ratios of phospho-AMPK/total AMPK and phospho-MEF2A/total MEF2A were significantly increased in the PCOS-MTF compared with the PCOS h-Ins group (P < 0.05). The RT-PCR experiments showed lower levels of GLUT4 mRNA transcripts in PCOS h-Ins compared with PCOS-MTF-treated group (P < 0.05), the protein levels of GLUT4 were decreased in a similar way. LIMITATIONS, REASONS FOR CAUTION: The limited number of patients included in this study who presented large clinical variability. Therefore, it would be necessary to recruit a greater number of patients to minimize our data dispersion in order to prove the clinical benefits of metformin described by others. WIDER IMPLICATIONS OF THE FINDINGS: Since the insulin sensitizer metformin increases the expression of the GLUT4, it may improve endometrial physiology in PCOS patients and, therefore, promote better reproductive outcomes. These results suggest that in PCOS patients, metformin may act directly at the endometrial level and decrease insulin resistance condition by increasing the expression of GLUT4 and, in this way, indirectly restore endometrial function. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (grant number 1095127 to M.V.). None of the authors has any conflict of interest to declare.

Protein expression of PKCZ (Protein Kinase C Zeta), Munc18c, and Syntaxin-4 in the insulin pathway in endometria of patients with polycystic ovary syndrome (PCOS).

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic disorder commonly associated with insulin resistance (IR). Previous studies indicate about the expression of molecules involved in the insulin pathway in endometria of women with PCOS-IR. Therefore, the aim of the present study was to evaluate the effect of insulin and testosterone in the expression of these proteins in the endometria and immortal endometrial stromal cell line (T-HESCs). METHODS: We examined the protein levels of Munc18c, PKC zeta, phospho-PKC Zeta, and Syntaxin-4. Protein levels were assessed by Western Blot and/or immunohistochemistry in proliferative endometria (NPE = 6) and in PCOS endometria with insulin resistance (PCOSE-IR = 6). We also evaluated whether high concentrations of insulin (100 nM) and/or testosterone (100 nM), during a 24 h stimulatory period, affected the expression of these proteins in an immortal endometrial stromal cell line (T-HESCs). Once stimulated, proteins were extracted from cells and were assessed by Western Blot analysis. Immunocytochemistry was performed to detect AR in T-HESC cells. RESULTS: Western Blot data showed decreased expression (p < 0,05) of Munc18c and phospho-PKC Zeta in PCOS-IR endometria (PCOSE-IR) with respect to the control (NPE). In the in vitro study, Western Blot analysis showed decreased levels of Munc18c, PKC Zeta and phospho-PKC Zeta with the different hormonal treatments when compared to the control condition (no hormonal stimulation) (p < 0,05). The AR was present in the endometrial stromal cell line (T-HESC). CONCLUSION: The conditions of hyperinsulinism and hyperandrogenism present in PCOS-IR patients modulate the expression and/or phosphorylation of the proteins involved in the insulin pathway at the endometrial level. These data extend to the T-HESCs cells results, where insulin and testosterone exert an effect on both the expression and phosphorylation of proteins present in the pathway.

Linfoma testicular: incidencia, descripción epidemiológica y manejo durante el período 2005-2011 / Testicular lymphoma: incidence, epidemiologic description and management during the 2005-2011 period

El linfoma testicular es una patología infrecuente, correspondiendo al 9 por ciento de los cánceres testiculares, presentándose más frecuentemente entre los 60 a 80 años (25-50 por ciento). La presentación clínica más frecuente es el aumento de volumen unilateral e indo/oro. El tipo histológico más común es linfoma difuso de células grandes B (60-90 por ciento). La orquidectomía radical asociada a quimioterapia y radioterapia es la primera línea de tratamiento para los pacientes con enfermedad limitada. Material y método: Estudio retrospectivo descriptivo. Se revisó y filtró la lista de pacientes ingresados al SIGGES como tumor testicular entre enero 2005 a abril 2011. De los pacientes con diagnóstico histológico e inmunohistoquímico compatible, se registraron las características epidemiológicas, estudio, manejo y sobrevida. Posteriormente se realizó un análisis de la base de datos con el programa estadístico SPSS 13. 0. Resultados: De un total de 299 pacientes con el diagnóstico histológico de cáncer testicular, 8 pacientes fueron diagnosticados como linfoma testicular confirmado por histología e inmunohistoquímica. El promedio y mediana de edad fue 52 años y 63 años (18-73) respectivamente. Tres casos (37,5 por ciento) correspondieron a presentaciones secundarias. En 6 de los casos (75 por ciento) el testículo afectado fue el derecho. Histológicamente, el 63 por ciento correspondió a Linfoma difuso de células grande B. Clínicamente, el todos los casos se presentaron con aumento de volumen y con marcadores en rango normal. En 7 casos (8 7, 5 por ciento) el diagnóstico y manejo inicial fue mediante orquidectomía radical, y en un caso por biopsia testicular, con orquidectomía posterior 3 casos presentaron diseminación...

esticular lymphoma is a rare disease, happening in 9 percent of testicular cancers, most commonly between the ages 60 to 80 years (25 percent-50 percent). The most common presentation is unilateral indolent testicular growth. Histology shows a diffuse big B cell lymphoma in most of the cases (60 percent-90 percent). Radical orchiectomy, chemotherapy and radiation are the first line therapy for patients with limited disease. Materials and methods: Retrospective clinical study. We included and filtered the SIGGES list of patients admitted for Testicular Tumor from January 2005 to April 2011. Patients with a compatible diagnosis were analyzed, using SPSS 13.0® as statistical software. Result: Of a total number of 299 testicular cancer patients 8 presented with a histological and inmunnohistochemical testicular lymphoma. Mean age was 52 years and the median 63 years (18-73). ln three cases (37.5 percent) it was a secondary localization. ln 6 cases ( 75 percent) the affected testicle was the right one. 63 percent corresponded to a diffuse big cell B cell Lymphoma. All patients presented normal tumor markers. ln 7 (87,5 percent) cases the initial treatment was radical orchiectomy in one patient the diagnosis was don through a testicular biopsy, and the orchidectomy was differed. 3 cases presented dissemination. In 7 patients adjuvant chemotherapy was performed. Mortal/ty was 38 percent with a 1 7-month follow-up. Conclusion: Testicular lymphoma is a rare condition with bad prognosis. Histology is fundamental for treatment, an in this sense inmunohystochemcal analysis is especially helpful...