The role of E-Cadherin expression in primary site of breast cancer.

PURPOSE: The tumour's ability to metastasize is the major cause for fatal outcomes in cancer diseases. In breast cancer, aberrant E-Cadherin expression has been linked to invasiveness and poor prognosis. METHOD: We assessed expression of E-Cadherin by immunohistochemistry in primary tumour tissue from 125 female breast cancer patients. Staining intensities were analysed using the immunoreactive score (IRS). We investigated E-Cadherin expression and its associations with clinicopathological parameters (age, tumour size, lymph node status, grade, hormone receptors, Her2 Status) as well as with recurrence and survival. RESULTS: Increased, rather than aberrant E-Cadherin expression was found and was associated with poor outcome (p = 0.046). Our data show an association between elevated E-Cadherin in primary tumour tissue and an unfavourable negative prognosis in patients. CONCLUSION: This association was somehow unexpected as loss of E-Cadherin has long been regarded as a prerequisite for development of invasiveness and metastases. Our findings support the notion that E-Cadherin promotes, rather than suppresses, development of metastasis and invasiveness.

Regulation of Epigenetic Modifications in the Placenta during Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells.

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

The role of EP-2 receptor expression in cervical intraepithelial neoplasia.

Prostaglandin induced signalling is involved in different cancers. As previously described, the EP3 receptor expression decreases with increasing stage of cervical intraepithelial lesions (CIN). In addition, in cervical cancer EP3 is an independent prognosticator for overall survival and correlates with FIGO stages. Currently the role of Prostaglandin 2 receptor 2 (EP2) in CIN is unknown. The aim of this study was to analyse the expression of EP2 for potential prognostic value for patients with cervical dysplasia. EP2 expression was analysed by immunohistochemistry in 33 patient samples (CIN1-3) using the immune-reactivity scoring system (IRS). Expression levels were correlated with clinical outcome to analyse prognostic relevance in patients with CIN2. Data analysis was performed using non parametric Kruskal-Wallis and Spearman rank sum test. Cytoplasmic expression levels of EP2 correlated significantly (p < 0.001) with different grades of cervical dysplasia. Median EP2-IRS in CIN1 was 2 (n = 8), 3 in CIN2 (n = 9) and 6 in CIN3 (n = 16). Comparing regressive (n = 3, median IRS = 2) to progressive (n = 6, median IRS = 4) CIN2 cases the median IRS differed significantly (p = 0.017). Staining intensity (p = 0.009) and IRS (p = 0.005) of EP2 and EP3 correlate inversely. EP2 expression level significantly increases with higher grade of CIN and could qualify as a potential prognostic marker for the regressive or progressive course in CIN2 lesions. These findings emphasize the significant role of PGE2 signalling in CIN and could help to identify targets for future therapies.

Delayed interval delivery in multiple gestations: the Munich experience.

OBJECTIVE: To evaluate delayed interval deliveries in multiple gestations in regard of delayed interval and neonatal survival and to provide a protocol. METHODS: Data of multiple pregnancies with delayed interval delivery at a tertiary maternity unit between 2002 and 2017 were collected. Contraindications for evaluation of a delay of the delivery of the remaining child were: severe maternal blood loss, poor maternal general condition, preeclampsia, placental abruption, fetal distress, serious congenital malformations of the remaining child, chorioamnionitis, and premature rupture of membranes of the second fetus. A total of 14 cases was included in this retrospective monocentric analysis. RESULTS: The cohort comprised nine twin and five triplet pregnancies. Mean gestational age at delivery of the first fetus was 21 + 6 and 26 + 0 of the retained fetus, respectively. The earliest delivery of the first fetus was at 15 + 2 weeks. The mean interval of the delay was 29.3 days (2-82 days). Mortality of the first fetuses was 53.3%, while it was 17.6% for the retained fetuses. Maternal outcome was good in general: two cases of major blood loss occurred with the necessity of a blood transfusion. CONCLUSION: Delayed interval delivery is a reasonable approach in cases of an imminent preterm birth in multiple gestations which can be performed with a good fetal outcome and limited maternal risks. The situation when this procedure may be an option always comes unexpected. Therefore, the team of perinatologists should keep it in mind as one potential therapeutic approach. In addition, a standard protocol for the procedure should be established in the perinatal center.

EP3 (prostaglandin E2 receptor 3) expression is a prognostic factor for progression-free and overall survival in sporadic breast cancer.

BACKGROUND: In various cancers, overexpression of cyclooxygenase (COX)-2 and elevated prostaglandin (PG) E2 synthesis have been associated with tumor development and progression. The potential of COX-2 inhibitors in cancer prevention and treatment has been shown repeatedly; however, their clinical use is limited due to toxicity. PGE2 signals via EP receptors 1-4, whose functions are analyzed in current research in search for targeted anti-PG therapies. EP2 and EP4 rather promote tumorigenesis, while the role of EP3, especially in breast cancer, is not yet clear and both pro- and anti-tumorigenic effects have been described. Our study evaluates EP3 receptor expression in sporadic breast cancer and its association with clinicopathological parameters, progression-free and overall survival. METHODS: Two hundred eighty-nine sporadic breast cancer samples without primary distant metastasis were immunohistochemically analyzed for EP3 receptor expression. Tissue was stained with primary anti-EP3-antibodies. Immunoreactivity was quantified by the immunoreactivity-score (IRS); samples with an IRS ≥ 2 scored as EP3 positive. Chi-squared and Mann-Whitney-U test were used for comparison of data; Kaplan-Meier estimates and Cox-regression were used for survival analyses. RESULTS: EP3 receptor was expressed in 205 of 289 samples analyzed (70.9%). EP3 receptor expression was not associated with clinicopathological parameters (e. g. tumor size, hormone receptors, lymph node status). Kaplan-Meier estimates showed a significant association of EP3 positivity with improved progression-free survival (p = 0.002) and improved overall survival (p = 0.001) after up to 10 years. Cox regression analysis confirmed EP3 positivity as a significant prognostic factor even when other known prognosticators were accounted for. CONCLUSIONS: In sporadic breast cancer, EP3 receptor expression is not significantly associated with clinicopathological parameters but is a significant prognostic factor for improved progression-free and overall survival. However, the functional aspects of EP3 receptor in breast cancer and the way how EP3 may oppose the pro-tumorigenic effects of PGE2 elevation and COX-2 overexpression are not fully understood so far. Further studies aiming at identification of the factors regulated by EP3 are necessary to evaluate the possibility of targeting EP3 in future anti-tumor therapy in breast cancer.

Evaluation of an interdisciplinary palliative care inhouse training for professionals in gynecological oncology.

PURPOSE: The aim of this study was to evaluate the effect of a pilot interdisciplinary inhouse training in palliative care (PC) for gynecological oncologists. METHODS: Competencies of participants from a gynecological university department were evaluated taking part in an interdisciplinary PC course in a pre and post design. The multiprofessional course covered basic principles of PC, symptom management and communication taught by PC specialists. Competencies were evaluated using self-designed questionnaires before (ISPG-1), right after (ISPG-2), and 6 months after the training (ISPG-3) (inhouse seminar palliative care in gynecology: ISPG). RESULTS: 31 persons from the department of gynecology took part in the course, of which 27 answered the first questionnaire (seven nurses (26%), 19 doctors (71%), one profession not indicated (3%), median working experience in gynecological oncology: 5 years). Return rates were: ISPG-1 27/31 (87.1%), ISPG-2 20/31 (64.5%) and IPSG-3 14/31 (45.2%). A more positive attitude towards PC could be observed in the majority of participants after the course (ISPG-2 62%, ISPG-3 71%). They felt more competent in the care of palliative patients (46%). PC would be initiated earlier and the interaction with other disciplines was improved (ISPG-2 85%, ISPG-3 100%). The participants assessed a significant improvement of their skills in all palliative fields which were analyzed. CONCLUSION: PC inhouse training improves the understanding of PC and the interdisciplinary approach in the management of patients with advanced disease. It is a feasible and useful instrument to improve the competencies in generalist PC of specialists in gynecological oncology.

PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas.

PPARγ belongs to the group of nuclear receptors which is expressed in the trophoblast and together with other factors is responsible for the maintenance of pregnancy. Apart from that PPARγ is also a main factor for macrophage polarization. The aim of this study was to investigate the combined expression pattern and frequency of PPARγ under physiological circumstances and in spontaneous and recurrent miscarriages in the trophoblast and in maternal macrophages of the decidua. Human placental tissues of the first trimester (15 physiologic pregnancies, 15 spontaneous abortion and 16 recurrent miscarriage placentas) were analyzed for expression of the nuclear receptor PPARγ. Expression changes were evaluated by immunohistochemistry and real time PCR (RT-PCR) in trophoblast and in maternal macrophages of the decidua. Maternal macrophages were identified by double immunofluorescence using cluster of differentiation 68 (CD68) as marker for macrophages and further characterized regarding their M1/M2 polarization status. The intermediate villous trophoblast revealed a significantly lower PPARγ expression in spontaneous and recurrent abortion. Maternal macrophages express PPARγ. Their number is significantly enhanced in the decidua of spontaneous miscarriages whereas in recurrent miscarriages maternal macrophages seem to express PPARγ only in very few cases. PPARγ is associated with an M2 polarization state that is common for decidual macrophages. The lack of PPARγ in recurrent miscarriage decidual macrophages seems to be associated with a specific inflammatory response against the fetus.

Epigenetic changes occur in placentas of spontaneous and recurrent miscarriages.

BACKGROUND: In contrast to genetic abnormalities which are well known to be responsible for around 50 % of human miscarriages, there is very few data about epigenetic alterations in spontaneous and recurrent miscarriages (SM, RM). The aim of this study was to analyze the histone modification marks H3K9ac and H3K4me3 in SM and RM. METHODS: The abundance of histone modifications H3K4me3/H3K9ac was analyzed by western blot in frozen abortion material of SM and RM compared to a control group of legal pregnancy terminations. Further, to characterize placental tissue cells expressing H3K4me3/H3K9ac immunohistochemistry (IHC) and immunofluorescence was performed in 20 SM, 19 RM and 26 controls. RESULTS: The western blot data showed a tendency to an overall reduction of H3K4me3/H3K9ac, in the placental tissue of particularly SM. Further we differentiated between syncytiotrophoblast, cytotrophoblast and decidual cells and found a significant decrease of H3K4me3 in SM in cytotrophoblast cells and syncytial stroma. In RM H3K4me3 was downregulated exclusively in the syncytiotrophoblast. H3K9ac was reduced in SM and RM in all evaluated compartments, except from the syncytiotrophoblast. CONCLUSION: Our study showed an overall reduced histone modification of H3K4me3 and H3K9ac in the placental tissue of SM. Concerning RM, particularly the reduction of H3K9ac was detected in the placental tissue, indicating that RM group has distinct profile in epigenetic regulation. Whether these histone modifications are part of a possible pathophysiologic cascade during SM and RM or are merely indicating a defective placentation, cannot be concluded from this study.

Epigenetic modification via H3K4me3 and H3K9ac in human placenta is reduced in preeclampsia.

BACKROUND: Alterations of DNA accessibility and chromatin structure are associated with diseases. We aimed to investigate epigenetic modifications in preeclampsia (PE), a pregnancy-associated hypertonic disease. Specifically, we addressed histone modification proteins H3K9ac (acetylated lysine 9 of the histone H3) and H3K4me3 (trimethylated lysine 4 of the histone H3) in PE. METHODS: We analyzed expression of histone proteins H3K4me3 and H3K9ac in 32 PE and 32 control placentas by immunohistochemistry. Further, we carried out confirmatory western blot analysis of respective proteins in 6 representative placentas. We then applied regression models with additional adjustment for potential confounders. RESULTS: Expression of H3K4me3 and H3K9ac is reduced in PE placentas as demonstrated by immunohistochemical stainings and western blot. There are no differences between female and male fetuses in the presence of these histone modifications. H3K4me3 positively correlated with maternal age (r = 0.444, p = 0.034). CONCLUSION: Expression of the placental histone proteins H3K4me3 and H3K9ac is reduced in PE, and independent of fetal gender. Our study underlines the involvement of epigenetic changes in the placenta of women suffering from PE.

Regulation of LCoR and RIP140 expression in cervical intraepithelial neoplasia and correlation with CIN progression and dedifferentiation.

PURPOSE: Ligand-dependent corepressor (LCoR) and receptor-interacting protein 140 (RIP140/NRIP1) play an important role in the regulation of multiple oncogenic signaling pathways and the development of cancer. LCoR and RIP140 form a nuclear complex in breast cancer cells and are of prognostic value in further prostate and cervical cancer. The purpose of this study was to analyze the regulation of these proteins in the development of cervical intraepithelial neoplasia (CIN I-III). METHODS: Immunohistochemical analysis was obtained to quantify RIP140 and LCoR expression in formalin-fixed paraffin embedded tissue sections of cervical intraepithelial neoplasia samples. Tissue (n = 94) was collected from patients treated in the Department of Gynecology and Obstetrics, Ludwig-Maximilians-University of Munich, Germany, between 2002 and 2014. Correlations of expression levels with clinical outcome were carried out to assess for prognostic relevance in patients with CIN2 progression. Kruskal-Wallis test and Mann-Whitney U test were used for data analysis. RESULTS: Nuclear LCoR overexpression correlates significantly with CIN II progression. Nuclear RIP140 expression significantly increases and nuclear LCoR expression decreases with higher grading of cervical intraepithelial neoplasia. Cytoplasmic RIP140 expression is significantly higher in CIN III than in CIN I or CIN II. CONCLUSION: A decrease of nuclear LCoR expression in line with an increase of dedifferentiation of CIN can be observed. Nuclear LCoR overexpression correlates with CIN II progression indicating a prognostic value of LCoR in cervical intraepithelial neoplasia. Nuclear and cytoplasmic RIP140 expression increases significantly with higher grading of cervical intraepithelial neoplasia underlining its potential role in the development of pre-cancerous lesions. These findings support the relevance of LCoR and RIP140 in the tumorigenesis indicating a possible role of LCoR and RIP140 as targets for novel therapeutic approaches in cervical intraepithelial neoplasia and cervical cancer.

EP3 receptor antagonist L798,106 reduces proliferation and migration of SK-BR-3 breast cancer cells.

Purpose: COX-2 overexpression and elevated levels of prostaglandin E2 (PGE2) play an important role in breast cancer carcinogenesis. Recently, expression of the PGE2 receptor EP3 has been shown to be a positive prognostic factor in breast cancer. This study analyzes the functional aspects of targeting EP3 in breast cancer cell lines. Material and methods: EP3 and EP1 expressions were determined in five breast cancer cell lines on the mRNA- and the protein-level. The selected cell lines were subsequently stimulated for 24-72 hrs with 10-1,000 nM of PGE2, the EP1/EP3 agonist sulprostone and the EP3 antagonist L798,106. Cell proliferation was determined via BrdU-assay, migration via scratch assay, EP3, Gi-protein and p-ERK1/2 expressions via Western blot and cAMP concentrations via ELISA. The Mann-Whitney-U-test was used to test for statistical significance. Results: The cell lines T-47D (EP3 expression 77.7%) and SK-BR-3 (EP3 expression 48.7%) were chosen. EP3 antagonism reduced its expression on SK-BR-3 significantly, while no effect was observed on T-47D. The proliferation and migration of SK-BR-3 cells were significantly reduced due to treatment with the EP1/3 agonist, the EP3 antagonist or a combination of both. Neither agonism nor antagonism influenced cell proliferation or migration in T-47D. In SK-BR-3, EP3 antagonism showed a significant decrease in Gi-protein levels, an increase in cAMP levels, and no significant change in p-ERK1/2 expression. Conclusion: Antagonism of the EP3 receptor results in a reduced proliferation and migration of SK-BR-3 breast cancer cells, potentially mediated via a Gi-protein-cAMP pathway. The results suggest that EP3 plays a role in tumorigenesis. This is in accordance with the cell culture data of other gynecological tumors, but it is conflicting in so far, as positive EP3 expression is clinically a positive prognostic marker in breast cancer. Therefore, other factors may be important in explaining this contradiction.

Involvement of ILR4α and TLR4 in miscarriages.

BACKGROUND: The purpose of this study was to analyze the involvement of signaling via Interleukin-4-Receptor α (IL4Rα) and Toll like receptor (TLR) 4 at the fetomaternal interface in the process of early pregnancy. PATIENTS AND METHODS: Placenta specimens of 46 patients in early pregnancy were analyzed (normal pregnancy (n = 15), spontaneous (n = 15) and habitual abortion (n = 16)). TLR4 and IL4Rα were analyzed by immunohistochemistry, immunofluorescence and real time PCR. Statistical analysis was carried out using SPSS 23 and Microsoft Excel. RESULTS: IL4Rα could be detected in trophoblast cells of all groups. It was significantly downregulated in the syncytiotrophoblast of spontaneous and recurrent abortions (p = 0.001), and in decidual tissue of spontaneous abortions (p = 0.001). Expression of TLR4 was decreased in the intermediate villous trophoblast (IVT) and decidua of spontaneous abortions (p = 0.04 & 0.003, respectively). On mRNA level expression of IL4Rα and TLR4 was significantly decreased in the group of recurrent miscarriages (IL4Rα p = 0.002, TLR4 p = 0.004). CONCLUSION: This study contributes new findings to the understanding of the complex molecular interplay at the fetomaternal interface in normal pregnancy and miscarriages. For the first time signaling via IL4Rα being involved at the very beginning of the generation of new life could demonstrated. Moreover, new evidence was provided regarding TLR4 playing a pivotal role in early pregnancy.

A randomized trial comparing limited-excision conisation to Large Loop Excision of the Transformation Zone (LLETZ) in cervical dysplasia patients.

OBJECTIVE: To show noninferiority of a limited-excision (resection of the dysplastic lesion only) vs. classical Large Loop Excision of the Transformation Zone (LLETZ). METHODS: In this prospective, randomized, multicenter trial, women with human papillomavirus (HPV) positive cervical intraepithelial neoplasia grade 3 were randomized into two groups (1:1). Primary outcome was the rate of negative HPV tests after 6 months, secondary outcomes included cone size, complete resection rates as well as cytological and histological results after 6 and 12 months. A sample size of 1,000 was calculated to show noninferiority of the limited-excision compared to the LLETZ group using a noninferiority margin of 5%. Enrollment was stopped after 100 patients due to slow accrual. RESULTS: Patients in the limited-excision group did not show a lower number of negative HPV tests (78% [LLETZ]-80% [limited-excision]=-2%; 90% confidence interval=-15%, 12%). The limited-excision resulted in a substantially lower cone size (LLETZ: 1.97 mL vs. limited-excision: 1.02 mL; p<0.001) but higher numbers of involved margins (LLETZ: 8% vs. limited-excision: 20%). Although postoperative cytological results slightly differed, histological results were similar in both groups. One limited-excision patient received immediate re-conisation, whereas one patient in each group was scheduled for re-conisation after 6 months. CONCLUSION: The limited-excision could represent a promising option to reduce the surgical extent of conisations while maintaining oncological safety. The trial was not sufficiently powered to reach statistical significance due to early termination. Nevertheless, the study provides important insights in the feasibility of a limited-excision and could serve as a pilot study for future trials. TRIAL REGISTRATION: German Clinical Trials Register Identifier: DRKS00006169.

The role of EP3-receptor expression in cervical dysplasia.

PURPOSE: Prostaglandin-mediated inflammatory reactions play a major role in different cancers. Prostaglandin E2-receptor 3 (EP3) expression correlates with FIGO stages in cervical cancer and has been shown to be an independent prognostic factor for overall survival. EP3 expression levels in cervical intraepithelial neoplasia (CIN) as the precursor lesion of cervical cancer are currently unknown. METHODS: EP3 expression was analyzed by immunohistochemistry in 124 patient samples (CIN 1-3 and healthy controls) using the IR-scoring system. Expression levels were correlated with clinical outcome to assess for prognostic relevance in patients with CIN 2. Data analysis was performed using Kruskal-Wallis and Mann-Whitney U test. RESULTS: EP3 expression levels significantly correlated with different grades of cervical dysplasia. Median EP3-IRS in healthy cervical tissue was 12 (n = 13) compared to 9 in CIN 1 (n = 38; p = 0.031 vs. healthy control), 6 in CIN 2 (n = 45; p < 0.001 vs. CIN 1) and 4 in CIN 3 (n = 28, p = 0.008 vs. CIN 2). The percentage of EP3 expressing cells in CIN 2 lesions was significantly lower in progressive than in regressive cases (mean percentage of EP3 positive cells in progress: 3.8%, n = 18; in regress: 9.3%, n = 20; p = 0.040). CONCLUSION: EP3 expression significantly decreases with higher grades of cervical intraepithelial neoplasia-which is in line with published IR scores in cervical cancer patients-and seems to be a prognostic marker for regression or progression of CIN 2 lesions. Our findings support the importance of the prostanoid pathway in cervical cancer and could help to identify targets for future therapies.

Detection of cervical intraepithelial neoplasia by using optical coherence tomography in combination with microscopy.

Optical coherence tomography (OCT) is a noninvasive high-resolution imaging technique that permits the detection of cancerous and precancerous lesions of the uterine cervix. The purpose of this study was to evaluate a new system that integrates an OCT device into a microscope. OCT images were taken from loop electrosurgical excision procedure (LEEP) specimens under microscopic guidance. The images were blinded with respect to their origin within the microscopic image and analyzed independently by two investigators using initially defined criteria and later compared to the corresponding histology. Sensitivity and specificity were calculated with respect to the correct identification of high-grade squamous intraepithelial lesions (HSIL). The interinvestigator agreement was assessed by using Cohen's kappa statistics. About 160 OCT images were obtained from 20 LEEP specimens. Sixty randomly chosen images were used to define reproducible criteria for evaluation. The assessment of the remaining 100 images showed a sensitivity of 88% (second investigator 84%) and a specificity of 69% (65%) in detecting HSIL. Surgical microscopy-guided OCT appears to be a promising technique for immediate assessment of microanatomical changes. In the gynecological setting, the combination of OCT with a colposcope may improve the detection of high-grade squamous intraepithelial lesions.