M.A. Arbós

ERM/ETV5 Up-regulation Plays a Role during Myometrial Infiltration through Matrix Metalloproteinase-2 Activation in Endometrial Cancer

We have described recently the Ets family transcription factor, ERM/ETV5, specifically up-regulated in endometrioid endometrial carcinoma (EEC) and associated with myometrial infiltration. Ets family members have been correlated to tumor progression by up-regulating the expression of matrix-degrading proteases. In the present study, we investigated the possibility that in EEC, ERM/ETV5 may act by inducing the expression of genes involved in extracellular matrix remodeling. Unraveling the molecular events associated with the initiation of tumor invasion would represent an obvious improvement for EEC patients. The overexpression of ERM/ETV5 induced scattering in the endometrial cancer cell line Hec-1A, correlating to increased matrix metalloproteinase-2 (MMP-2) gelatinase activity. Both chromatin immunoprecipitation and reversion experiments with RNA interference and specific MMP-2 inhibitor showed a functional link between ERM/ETV5 overexpression and MMP-2 activation. The increased MMP-2 activity associated with overexpressed ERM/ETV5 in a mouse model conferred invasive capacity to endometrial tumors. Orthotopically implanted overexpressing ERM/ETV5 tumors presented a more aggressive and infiltrative pattern of myometrial invasion. Finally, the specific localization of ERM/ETV5 and MMP-2 at the invasive front of myometrial infiltrating human endometrial carcinomas further reinforced the hypothesis of a role for ERM/ETV5 in the early steps of endometrial dissemination. Taken together, these results lead us to propose that in EEC, ERM/ETV5 acts through MMP-2 gelatinolytic activity to confer invasive capabilities, associated with an initial switch to myometrial infiltration. They also postulate ERM/ETV5 as a valuable marker for patient stratification and a transcription pathway that should be evaluated for therapies specifically targeting the initial steps of EEC dissemination.

Antiproliferative and apoptotic effects of the herbal agent Pygeum africanum on cultured prostate stromal cells from patients with benign prostatic hyperplasia (BPH).

Previous reports show that the herbal agent Pygeum africanum (PA) used to treat benign prostatic hyperplasia (BPH) inhibits proliferation of prostate stromal cells from BPH tissues. To determine underlying mechanisms, we compared proliferative and apoptotic responses to PA between BPH and non‐BPH prostate stromal cells with a focus on the specific reaction displayed by stromal cell subsets. An interaction of PA with growth factors and hormones was also investigated.

MMPs/TIMPs and inflammatory signalling de-regulation in human incisional hernia tissues.

Background: Incisional hernia is a common and important complication of laparotomies. Epidemiological studies allude to an underlying biological cause, at least in a subset of population. Interest has mainly focused on abnormal collagen metabolism. However, the role played by other determinants of extracellular matrix (ECM) composition is unknown. To date, there are few laboratory studies investigating the importance of biological factors contributing to incisional hernia development. We performed a descriptive tissue‐based analysis to elucidate the possible relevance of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in association with local cytokine induction in human incisional hernia tissues. The expression profiles of MMPs, TIMPs and pro‐inflammatory cytokine signalling were investigated in aponeurosis and skeletal muscle specimens taken intraoperatively from incisional hernia (n= 10) and control (n= 10) patients. Semiquantitative RT‐PCR, zymography and immunoblotting analyses were done. Incisional hernia samples displayed alterations in the microstructure and loss of ECM, as assessed by histological analyses. Moreover, incisional hernia tissues showed increased MMP/TIMP ratios and de‐regulated inflammatory signalling (tumor necrosis factor [TNFA] and interleukin [IL]‐6 tended to increase, whereas aponeurosis TNFA receptors decreased). The changes were tissue‐specific and were detectable at the mRNA and/or protein level. Statistical analyses showed several associations between individual MMPs, TIMPs, interstitial collagens and inflammatory markers. The increment of MMPs in the absence of a counterbalance by TIMPs, together with an ongoing de‐regulated inflammatory signalling, may contribute in inducing a functional defect of the ECM network by post‐translational mechanisms, which may trigger abdominal wall tissue loss and eventual rupture. The notable TIMP3 protein down‐regulation in incisional hernia fascia may be of pathophysiological significance. We conclude that this study may help to pinpoint novel hypotheses of pathogenesis that can lead to a better understanding of the disease and ultimately to improvement in current therapeutic approaches.

Exploring the clinical validity of borderline personality disorder components.

Borderline personality disorder (BPD) is recognized as a complex syndrome, resulting in a heterogeneous diagnostic category. Besides the characteristics of the disorder itself, comorbid disorders play an important role in this complexity. The aim of the study is to analyze the clinical validity of 3 components for BPD Diagnostic and Statistical Manual of Mental Disorders criteria--called affective dysregulation, behavioral dysregulation, and disturbed relatedness--investigating differences in patterns of comorbidity. For this purpose, 365 patients with suspected BPD were included in the study. To test our hypothesis, patients were classified into 5 clusters using a K-cluster analysis to study the clinical validity of the 3 components based on the 3-factor model of BPD. Differences in comorbidity, previous suicide attempts, and self-harm behaviors among the defined clusters were analyzed. Between-cluster differences were observed for Axis I and Axis II disorders as well as in the frequency of suicide attempts and in self-harm behaviors. The study of BPD based on the 3 components seems to be more useful than the study of BPD as a unitary construct to help further our understanding of this complex disorder. In the present study, the 3 BPD components have allowed us to analyze the complex comorbidity of BPD patients. This solution could be considered an interesting way to clarify BPD etiology, diagnosis, and treatment efficacy.

The impact of KRAS mutations on VEGF-A production and tumour vascular network

BackgroundThe malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile.MethodsCodon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results.ResultsOur results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter.ConclusionSubtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis.

PREBIOUS trial: A multicenter randomized controlled trial of PREventive midline laparotomy closure with a BIOabsorbable mesh for the prevention of incisional hernia: Rationale and design

BACKGROUND Development of an incisional hernia is one of the most frequent complications of midline laparotomies requiring reoperation. This paper presents the rationale, design, and study protocol for a randomized controlled trial, the aim of which is to evaluate the efficacy and safety of prophylactically placing a bioabsorbable synthetic mesh for reinforcement of a midline fascial closure. METHODS The PREBIOUS trial (PREventive midline laparotomy closure with a BIOabsorbable mesh) is a multicenter randomized controlled trial in which adult patients undergoing elective or urgent open abdominal operations through a midline laparotomy incision are assigned to one of two groups based on the laparotomy closure procedure: an intervention group in which a continuous polydioxanone (PDS) suture is reinforced with a commercially available GORE® BIO-A® Tissue Reinforcement prosthesis (W.L. Gore & Associates, Flagstaff, AZ, USA), or a control group with continuous PDS suture only. Both groups are followed over 6 months. OUTCOMES The primary outcome is the appearance of incisional hernias assessed by physical examination at clinical visits and radiologically (CT scan) performed at the end of follow-up. Secondary outcomes are the rate of complications, mainly infection, hematoma, burst abdomen, pain, and reoperation. The PREBIOUS trial has the potential to demonstrate that suture plus prosthetic mesh insertion for routine midline laparotomy closure is effective in preventing incisional hernias after open abdominal surgery, to avoid the effects on those affected, such as poor cosmesis, social embarrassment, or impaired quality of life, and to save costs potentially associated with incisional hernia surgical repair.

Significado clínico de la atrofia proliferativa inflamatoria en la biopsia prostática

INTRODUCTION Proliferative inflammatory atrophy (PIA) is a frequently observed lesion in prostate biopsies and some authors have postulated its involvement in prostate carcinogenesis. However, the mechanisms that would permit its neoplastic transformation and the clinical significance of its finding in a prostate biopsy is currently not well known. OBJECTIVE To analyze the characteristics of the PIA lesion, its possible role in prostate carcinogenesis and its relation with the tumor aggressiveness. MATERIAL AND METHOD A systematic review was made of the literature in PubMed with the terms «proliferative inflammatory atrophy» or «PIA» and «prostate.» The most important findings are summarized in accordance with the study objective. RESULTS PIA seems to be involved in prostate carcinogenesis. This hypothesis is based on its frequent association to cancer lesions (CaP) and on some genetic alterations that are common to the high grade prostatic intraepithelial neoplasia (HGPIN) and to the CaP, fundamentally deficit in GSTP1 expression and overexpression of AGR2. Currently, there are no epidemiological studies that evaluate the incidence of PIA or its association with HGPIN and CaP. Only one study, carried out by our group, has determined the global incidence of PIA in 30% of the prostate biopsies, a lower association to CaP than the HGPIN lesion and an association between PIA and tumors of lower and insignificant grade. CONCLUSIONS PIA shares genetic alterations with HGPIN and CaP. Currently, there is no epidemiologic evidence to consider that the PIA is associated to a greater incidence of CaP and the genetic and epidemiological data available suggest its association to not very aggressive tumors.

Apoptosis-Like Cell Death Induction and Aberrant Fibroblast Properties in Human Incisional Hernia Fascia

Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo.

ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer

Endometrial cancer (EC) is the sixth deadliest cancer in women. The depth of myometrial invasion is one of the most important prognostic factors, being directly associated with tumor recurrence and mortality. In this study, ALCAM, a previously described marker of EC recurrence, was studied by immunohistochemistry at the superficial and the invasive tumor areas from 116 EC patients with different degree of myometrial invasion and related to a set of relevant epithelial and mesenchymal markers. ALCAM expression presented a heterogeneous functionality depending on its localization, it correlated with epithelial markers (E-cadherin/β-catenin) at the superficial area, and with mesenchymal markers at the invasive front (COX-2, SNAIL, ETV5, and MMP-9). At the invasive front, ALCAM-negativity was an independent marker of myometrial invasion. This negativity, together with an increase of soluble ALCAM in uterine aspirates from patients with an invasive EC, and its positive correlation with MMP-9 levels, suggested that ALCAM shedding by MMP-9 occurs at the invasive front. In vivo and in vitro models of invasive EC were generated by ETV5-overexpression. In those, we demonstrated that ALCAM shedding was related to a more invasive pattern and that full-ALCAM recovery reverted most of the ETV5-cells mesenchymal abilities, partially through a p-ERK dependent-manner.

MP41-07 PROLIFERATIVE INFLAMMATORY ATROPHY IS ASSOCIATED TO LESS AGGRESSIVE AND INSIGNIFICANT TUMORS IN RADICAL PROSTATECTOMY SPECIMENS

INTRODUCTION AND OBJECTIVES: Proliferative inflammatory atrophy (PIA) has been proposed to be involved in prostate carcinogenesis by suggesting that it may rise to prostatic carcinoma (PCa) either directly or indirectly by first developing into high grade prostatic intraepithelial neoplasm (HGPIN). We have studied the PIA genetic signature 2 and more recently we have learned that PIA incidence in prostatic biopsies is around 30%, and it is associated to lower PCa detection and less aggressive tumors . The objectives of this study were to assess the incidence of PIA lesion in radical prostatectomy (RP) specimens and to confirm the hypothesis that PIA is associated to less aggressive tumors. METHODS: A retrospective study conducted in 200 consecutive patients subjected to RP was done. A single pathologist identified PIA in all specimens and this finding was related with: age, serum PSA, prostate volume, Gleason score, pathologic stage, percentage of tumor in the gland, maximal length of index tumor, perineural invasion, multifocality, bilaterality, insignificant cancer, positive margins, and HGPIN. Mann-Witney U test and Chi-square Pearson test were used to compare data. RESULTS: PIA was present in 64 specimens (32%). Age, serum PSA and prostate volume were similar in patients with and without PIA. Respectively, high Gleason grade was present in 34.4% and 49.3%, p1⁄40.033; non-organ confine disease in 10.9% and 16.9%, p1⁄40.187; perineural invasion in 59.4 and 74.3%, p1⁄40.025; multifocal tumors in 59.4% and 74.3%, p1⁄40.001; bilateral tumors in 46.9% and 70.5%, p1⁄40.005; insignificant tumors in 17.2% and 6.6%, p1⁄40.022; HGPIN in 92.2% and 90.4%, p1⁄40.456. CONCLUSIONS: This study confirms that the incidence of PIA in RP specimens was similar to the one observed previously in prostatic biopsies (32% versus 30%). PIA was not associated to HGPIN. PIA was associated to many pathologic characteristics of less aggressive tumors. Of great clinical interest could be the finding that PIA is associated to unilaterial, unifocal, low grade, and insignificant tumors. 1 De Marzo et al. Am J Pathol 1999; 155: 1985-92. 2 Morote et al. Annual EAU Congress 2012; P-848. 3 Morote el al. Annual AUA Meeting 2013; P-2226.