Adrenomedullin Secreted by Melanoma Cells Promotes Melanoma Tumor Growth through Angiogenesis and Lymphangiogenesis.

INTRODUCTION: Metastatic melanoma is an aggressive tumor and can constitute a real therapeutic challenge despite the significant progress achieved with targeted therapies and immunotherapies, thus highlighting the need for the identification of new therapeutic targets. Adrenomedullin (AM) is a peptide with significant expression in multiple types of tumors and is multifunctional. AM impacts angiogenesis and tumor growth and binds to calcitonin receptor-like receptor/receptor activity-modifying protein 2 or 3 (CLR/RAMP2; CLR/RAMP3). METHODS: In vitro and in vivo studies were performed to determine the functional role of AM in melanoma growth and tumor-associated angiogenesis and lymphangiogenesis. RESULTS: In this study, AM and AM receptors were immunohistochemically localized in the tumoral compartment of melanoma tissue, suggesting that the AM system plays a role in melanoma growth. We used A375, SK-MEL-28, and MeWo cells, for which we demonstrate an expression of AM and its receptors; hypoxia induces the expression of AM in melanoma cells. The proliferation of A375 and SK-MEL-28 cells is decreased by anti-AM antibody (αAM) and anti-AMR antibodies (αAMR), supporting the fact that AM may function as a potent autocrine/paracrine growth factor for melanoma cells. Furthermore, migration and invasion of melanoma cells increased after treatment with AM and decreased after treatment with αAMR, thus indicating that melanoma cells are regulated by AM. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing melanoma cells, as demonstrated by reduced numbers of vessel structures, which suggests that AM is one of the melanoma cells-derived factors responsible for endothelial cell-like and pericyte recruitment in the construction of neovascularization. In vivo, αAMR therapy blocked angiogenesis and lymphangiogenesis and decreased proliferation in MeWo xenografts, thereby resulting in tumor regression. Histological examination of αAMR-treated tumors showed evidence of the disruption of tumor vascularity, with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. CONCLUSIONS: The expression of AM by melanoma cells promotes tumor growth and neovascularization by supplying/amplifying signals for neoangiogenesis and lymphangiogenesis.

Impact of antibiotic treatment for chronic endometritis on unexplained recurrent pregnancy loss.

INTRODUCTION: Recurrent Pregnancy Loss (RPL) affects about 1% of all couples and is likely to cause therapeutic vagrancy and psychological distress. Multiple origins can explain RPL, and recent studies suggest the influence of chronic endometritis. The aim of our study is to evaluate the impact of antibiotic treatment on obstetrical prognosis among patients consulting for RPL with isolated chronic endometritis. MATERIAL AND METHODS: We conducted a monocentric retrospective comparative study. Patients consulting for RPL, with normal etiologic examinations (except for chronic endometritis), were included. In the case of chronic endometritis, patients could receive antibiotic treatment (14 days of doxycycline and metronidazole). Pregnancy outcomes, collected one year after inclusion, were compared between 3 groups: patients without chronic endometritis, patients with treated chronic endometritis, patients with untreated chronic endometritis. Univariate and multivariate analyses were performed. RESULTS: 42 patients were included. 22 patients had chronic endometritis. Groups were comparable in terms of age, BMI, the number of miscarriages, tobacco consumption, AMH, and FSH levels on day 2. In multivariate analysis, a significant improvement of live birth rate was observed among patients treated for chronic endometritis, compared to the no endometritis group (OR 21.4 [1.93-236.70] p = 0.013) and the untreated endometritis group (OR 24.90 [1.64-376.93] p = 0.020). CONCLUSION: In our patients examined for RPL, the live birth rate was improved after treatment of chronic endometritis with 14-day antibiotic treatment in comparison to patients with untreated chronic endometritis.

Mechanical characterisation of human ascending aorta dissection.

Mechanical characteristics of both the healthy ascending aorta and acute type A aortic dissection were investigated using in vitro biaxial tensile tests, in vivo measurements via transoesophageal echocardiography and histological characterisations. This combination of analysis at tissular, structural and microstructural levels highlighted the following: (i) a linear mechanical response for the dissected intimomedial flap and, conversely, nonlinear behaviour for both healthy and dissected ascending aorta; all showed anisotropy; (ii) a stiffer mechanical response in the longitudinal than in the circumferential direction for the healthy ascending aorta, consistent with the histological quantification of collagen and elastin fibre density; (iii) a link between dissection and ascending aorta stiffening, as revealed by biaxial tensile tests. This result was corroborated by in vivo measurements with stiffness index, ß, and Peterson modulus, Ep, higher for patients with dissection than for control patients. It was consistent with histological analysis on dissected samples showing elastin fibre dislocations, reduced elastin density and increased collagen density. To our knowledge, this is the first study to report biaxial tensile tests on the dissected intimomedial flap and in vivo stiffness measurements of acute type A dissection in humans.

Endovascular creation and validation of acute in vivo animal model for type A aortic dissection.

BACKGROUND: Animal modeling is a prerequisite for clinical transfer of new therapies. This study targets an acute in vivo animal model of type A dissection using endovascular approach with a view to test future stent grafts dedicated to this aortic segment. METHODS: Experiments were conducted on 13 swine. Two arterial accesses, femoral and percutaneous transapical, were required. Entry tear was created by endovascular instrumental means inserted through transapical access with either Outback catheter (group 1, n = 3) or EchoTip Endoscopic Ultrasound Needle (group 2, n = 10). Afterward, dissection extension was obtained in antegrade direction by looped guidewire technique, and, as often as possible, re-entry tear was created with either looped guidewire or Outback catheter. Finally, entry tear, dissected space, and re-entry tear when existing were dilated with 8-mm balloon. In our acute model, animals were euthanized at the end of the experiment day, and aortas were explanted for macroscopic and histologic examination. RESULTS: The model was successfully created in 10 out of 13 animals. In group 1, dissection was limited to arch with 23 mm average length and no possibility of achieving re-entry tear. One aortic perforation was observed. In group 2, dissection was extended up to descending thoracic or thoracoabdominal aorta, with 110 mm average length (range 40-165 mm), and re-entry tear was created in seven cases. Histologic examination confirmed the presence of intimo-medial flap. CONCLUSIONS: The present experiment validates a new type A dissection animal model, which morphologically reproduces human aortic dissection features. As such, it provides an advantageous basis for testing future stent grafts.

Is the perceived placebo effect comparable between adults and children? A meta-regression analysis.

BACKGROUND: A potential larger perceived placebo effect in children compared with adults could influence the detection of the treatment effect and the extrapolation of the treatment benefit from adults to children. This study aims to explore this potential difference, using a meta-epidemiological approach. METHODS: A systematic review of the literature was done to identify trials included in meta-analyses evaluating a drug intervention with separate data for adults and children. The standardized mean change and the proportion of responders (binary outcomes) were used to calculate the perceived placebo effect. A meta-regression analysis was conducted to test for the difference between adults and children of the perceived placebo effect. RESULTS: For binary outcomes, the perceived placebo effect was significantly more favorable in children compared with adults (ß = 0.13; P = 0.001). Parallel group trials (ß = -1.83; P < 0.001), subjective outcomes (ß = -0.76; P < 0.001), and the disease type significantly influenced the perceived placebo effect. CONCLUSION: The perceived placebo effect is different between adults and children for binary outcomes. This difference seems to be influenced by the design, the disease, and outcomes. Calibration of new studies for children should consider cautiously the placebo effect in children.

Correlation of cytotechnologists' parameters with their performance in rapid prescreening of papanicolaou smears.

BACKGROUND: Efficient quality control is essential to ensure high sensitivity of Papanicolaou (Pap) smears. For this purpose, rescreening of 10% random negative smears is increasingly felt to be ineffective. Rapid rescreening (RR) of all negative Pap smears is more practical and has received widespread acceptance, especially in Europe, although its sensitivity is difficult to monitor and its retrospective nature may influence the vigilance of the screeners. The method of rapid prescreening (RPS) overcomes these drawbacks because rapid review of Pap smears precedes full screening. METHODS: All routine conventional Pap smears (n = 8364) over 2 months underwent RPS by 12 cytotechnologists, followed by full screening. Data were analyzed to determine correlation between the RPS sensitivity of individual cytotechnologists and both their sensitivity in full screening and their years of experience as cytotechnologists. RESULTS: There was a striking variability in sensitivity (15.4%-72.7%) among the 12 screeners with an atypical squamous cells of undetermined significance (ASCUS) threshold. There was no correlation between RPS sensitivity of individual cytotechnologists with either their sensitivity in full screening or their years of experience as cytotechnologists. CONCLUSIONS: The skills required of a cytotechnologist for achieving a high sensitivity in RPS are apparently different from those of full screening and are independent of the sensitivity of the screeners at full screening or of the years of experience as cytotechnologists.

Rapid prescreening of Papanicolaou smears: a practical and efficient quality control strategy.

BACKGROUND: Efficient quality control (QC) is essential to ensure high sensitivity of Papanicolaou (Pap) smears. For this purpose, rescreening of 10% random negative smears is ineffective. Rapid rescreening (RR) of all negative Pap smears is more practical and has received widespread acceptance, especially in Europe, although its sensitivity is difficult to monitor and its retrospective nature may influence the vigilance of the screeners. The method of rapid prescreening (RPS) overcomes these drawbacks because rapid review of Pap smears occurs before routine full screening. METHODS: All routine conventional Pap smears over 2 months underwent RPS by 12 cytotechnologists. Approximately 30 seconds were allowed to prescreen each slide. The presence of abnormal cells (atypical squamous cells of undetermined significance [ASCUS] or above), infection or endometrial cells detected on RPS was documented. All slides subsequently underwent routine full screening. Results of both screening methods were compared. RESULTS: Of a total of 8364 Pap smears, 310 (3.7%) cases were categorized as abnormal after final diagnosis. Of those, 135 were also detected on RPS (sensitivity of 43.5%). Seventeen abnormal cases were detected only on RPS: these consisted of 13 ASCUS cases, 3 low-grade squamous intraepithelial lesions, and 1 high-grade squamous intraepithelial lesion. The sensitivity of RPS for infections and endometrial cells was 51.6% and 28.3%, respectively. Implementation of RPS did not significantly impact the work flow in our laboratory. CONCLUSIONS: RPS is an efficient and practical QC tool. It is a reliable method with which to monitor sensitivity and reduce the false-negative rate, and because it is done before finalizing the case, it allows for timely corrections to the diagnosis and avoids the need to amend reports.

Comparison of the prognosis indication of VEGFR-1 and VEGFR-2 and Tie2 receptor expression in breast carcinoma.

The degree of angiogenesis in breast cancer has previously been shown to be an indicator of prognosis, and tumor microvasculature is a candidate target for new antiangiogenic therapies. The aim of this study was to investigate the prognostic value of vascular endothelial growth factor (VEGF) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and Tie2/tek receptor tyrosine kinase in breast carcinoma. VEGF receptors and Tie2 expression was investigated using immunohistochemical assays with monoclonal antibodies on frozen sections in a series of 918 and 909 patients respectively. VEGFR-1 and VEGFR-2 and Tie2 were correlated with long-term (median, 11.3 years) patients' outcome. Univariate (Kaplan-Meier) analysis showed that VEGFR-1 positive tumor surface (cutoff = 5%) was significantly correlated with high metastasis risk (p=0.03) and relapse (p<0.01) in all patients, and in those with node negative tumors (p<0.001 and p<0.01 respectively), but not with overall survival. In contrast Tie2 positive tumor surface (cutoff = 7%) was significantly correlated with poor overall survival (p=0.025) and also with high metastasis risk particularly among node negative patients (p<0.01). Moreover, Tie2 immunoexpression was significantly predictive of relapse (p=0.003) in the node negative subgroup (p=0.02). In multivariate analysis (Cox model), VEGFR-1 and Tie2 immunoexpressions were identified as independent prognostic indicators. In contrast, univariate analysis showed that VEGFR-2 positive tumor surface (cutoff = 10%) was not correlated with survival or with metastasis and relapse risk. Our results suggest that VEGFR-1 and Tie2 immunohistochemical expression permits the identification of patients with poor outcome, and particularly node negative ones with a high risk for metastasis and relapse. VEGFR-1 and Tie2 immunodetection may also be considered as potential tools for selecting patients who could benefit in the future from specific antiangiogenic therapy interfering with VEGFR-1 and Tie2 activation pathways.

[Immunohistochemical expression of PTEN antigen: a new tool for diagnosis of early endometrial neoplasia]. / L'expression immunohistochimique de l'antigène PTEN: un nouvel outil diagnostique dans les néoplasies débutantes de l'endomètre.

We examined immunohistochemical PTEN protein expression in endometrial samples obtained from perimenopausal women with uterine bleeding in order to diagnose early endometrial neoplasia. Paraffin sections of endometrial samples (biopsies, endometrium resection, hysterectomy) were reviewed and tested for immunocytochemical PTEN expression by epithelial cells. Immunohistochemical studies were performed with the Ventana Benchmark device and the immunoperoxidase technique with monoclonal anti-PTEN (6H2.1/Cascade Biosciences). We studied 45 samples of proliferative endometria, 42 samples of atypical hyperplasia (neoplasia), and 55 samples of endometrial carcinomas (30 endometrioid, 5 serous papillary, 10 clear cell, 5 adenosquamous, and 5 MMMT). A strong positive PTEN immunoreaction was observed in all 45 normal proliferative endometria, 2/30 endometrioid carcinomas, and 23/25 non endometrioid carcinomas. In contrast, PTEN immunoexpression was negative or weak in 40/42 atypical hyperplasia and 28/30 endometrioid carcinomas. Negative or weak PTEN expression correlated with endometrial neoplasia (atypical proliferation) (p < 0.001). Thus (i) PTEN immunoexpression in endometrial paraffin sections is a new diagnostic tool, distinguishing PTEN-positive cyclic abnormal monoclonal proliferative endometrium (anovulatory, non atypical, simple and complex hyperplasia) from PTEN-negative (or decreased) endometrial neoplasia, and especially early endometrial neoplasia (atypical simple and complex hyperplasia, in situ carcinoma, superficial carcinoma with focal invasion). (ii) Given the lack of reproducibility of histological identification of atypical (precancerous invasive) and non atypical (precancerous non invasive) endometrial hyperplasia, the new criteria (PTEN-negative crowded glands, gland/stroma ratio > 55%, nuclear pleomorphism, in areas > 1 mm) recently proposed to diagnose early endometrial neoplasia appear to be clinically relevant.

Tumor neoangiogenesis by CD31 and CD105 expression evaluation in breast carcinoma tissue microarrays.

PURPOSE: The aim of the study was to evaluate CD31 and CD105 immunohistochemical expressions in tissue microarrays from 360 breast carcinomas. STUDY DESIGN: Computerized (ACIS/Chromavision) assisted image analysis was performed to compare immunoreactions in tissue microarrays with those in current paraffin and frozen sections. We also aimed to determine the CD105 and CD31 prognostic significance and relevance in routine practice by correlating results of immunodetections with patients' (n = 360) outcome (14.3-year follow-up). RESULTS: The results show (a) that in tissue microarrays, the CD31 and CD105 expression quantified by image analysis device did not correlate with the measurements assessed on routine paraffin sections; (b) that CD105 expression is endowed of a prognostic significance in paraffin sections in terms of overall survival (P < 0.01), whereas in contrast, CD31 on paraffin sections did not correlate with patients overall survival; (c) that semiquantitative analysis of CD105 expression correlated with the image analysis measurements in frozen sections (rho = 0.671, P < 0.01) and paraffin (rho = 0.824, P < 0.01) sections. However, paraffin sections were less immunostained than frozen ones. CONCLUSIONS: It is concluded (a) that CD105 may be suitable in paraffin sections to evaluated neoangiogenesis; and (b) that tissue microarrays are not suitable substrates for neoangiogenesis evaluation as a prognostic indicator in breast carcinomas, in contrast to current tissue sections.

[Prognostic significance of VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) in breast carcinoma]. / Valeur pronostique de l'expression des recepteurs du VEGF, VEGFR-1 (FLT-1) et VEGFR-2 (KDR/FLK-1) dans le cancer du sein.

OBJECTIVES: The aim of this study was to investigate the prognostic value of vascular endothelial growth factor (VEGF) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) in breast carcinoma. METHODS: VEGF receptor expression was investigated using immunohistochemical assays with monoclonal antibodies on frozen sections in a series of 918 patients and was correlated with prognostic parameters and with long-term follow-up (median, 11.3 years). VEGFR-1 and VEGFR-2 immunostained surface was evaluated in percentage of the total tumor specimen surface by light microscopy (x100). RESULTS: VEGFR-1 and VEGFR-2 were strongly expressed in endothelial cells within blood microvessels, and weakly in tumor cells. Univariate (Kaplan Meier) analysis showed that VEGFR-1 positive tumor surface (cut off=5%) was not correlated with survival, but was significantly correlated with high metastasis risk (p=0.03) and relapse (p=0.01) in all patients, and in those with node negative tumors (p=0.001 and p=0.01 respectively). In multivariate analysis (Cox model), VEGFR-1 expression was identified as an independent prognostic indicator. Univariate analysis showed that VEGFR-2 positive tumor surface (cut off=10%) was not correlated with survival or with metastasis risk and relapse. CONCLUSION: Our results show that VEGFR-1 immunohistochemical expression permits the identification of patients with poor outcome, particularly those with node negative tumors, with high risk of metastasis and relapse. VEGFR-1 immunodetection may further be considered as a potential tool for evaluating tumor agressiveness and therapeutic strategies in breast cancer.