DNA methylation-based classifier differentiates intrahepatic pancreato-biliary tumours.

BACKGROUND: Differentiating intrahepatic cholangiocarcinomas (iCCA) from hepatic metastases of pancreatic ductal adenocarcinoma (PAAD) is challenging. Both tumours have similar morphological and immunohistochemical pattern and share multiple driver mutations. We hypothesised that DNA methylation-based machine-learning algorithms may help perform this task. METHODS: We assembled genome-wide DNA methylation data for iCCA (n = 259), PAAD (n = 431), and normal bile duct (n = 70) from publicly available sources. We split this cohort into a reference (n = 399) and a validation set (n = 361). Using the reference cohort, we trained three machine learning models to differentiate between these entities. Furthermore, we validated the classifiers on the technical validation set and used an internal cohort (n = 72) to test our classifier. FINDINGS: On the validation cohort, the neural network, support vector machine, and the random forest classifiers reached accuracies of 97.68%, 95.62%, and 96.5%, respectively. Filtering by anomaly detection and thresholds improved the accuracy to 99.07% (37 samples excluded by filtering), 96.22% (17 samples excluded), and 100% (44 samples excluded) for the neural network, support vector machine and random forest, respectively. Because of best balance between accuracy and number of predictable cases we tested the neural network with applied filters on the in-house cohort, obtaining an accuracy of 95.45%. INTERPRETATION: We developed a classifier that can differentiate between iCCAs, intrahepatic metastases of a PAAD, and normal bile duct tissue with high accuracy. This tool can be used for improving the diagnosis of pancreato-biliary cancers of the liver. FUNDING: This work was supported by Berlin Institute of Health (JCS Program), DKTK Berlin (Young Investigator Grant 2022), German Research Foundation (493697503 and 314905040 - SFB/TRR 209 Liver Cancer B01), and German Cancer Aid (70113922).

Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

Comparative investigation of cell cycle and immunomodulatory genes in mucosal and cutaneous melanomas: Preliminary data suggest a potential promising clinical role for p16 and the PD-1/PD-L1 axis.

Mucosal melanomas arise from the mucosal lining of various organs. Their etiology is currently unknown and there are no tissue-based methods to differentiate it from cutaneous melanomas. Furthermore, prognostic and predictive markers (e.g. for immune checkpoint inhibition) are lacking. In this study, we aimed to assess the protein expression levels of cell cycle-associated proteins and immune checkpoint markers in a cohort of mucosal melanomas in comparison to cutaneous melanomas and evaluated the effect of potential regulatory mechanisms. We performed immunohistochemistry, DNA methylation analysis and copy number profiling of 47 mucosal and 28 cutaneous melanoma samples. Protein expression of CD117, Ki67 and p16 was higher in mucosal melanomas, while BCL2, Cyclin D1, PD-1 and PD-L1 were overexpressed in cutaneous melanomas. CDKN2A deletions were the most prevalent numeric chromosomal alterations in both mucosal and cutaneous melanoma and were associated with decreased p16 expression. KIT was frequently amplified in mucosal melanomas, but not associated with CD117 expression. On the other hand, amplification of CCND1 lead to Cyclin D1 overexpression. In mucosal melanoma patients high PD-1 expression and high PD-L1 promoter methylation levels were associated with improved survival. PD-L1 expression correlated with response to immune checkpoint inhibitor therapy in the combined group of melanoma patients. Mucosal and cutaneous melanomas show different expression levels of cell cycle-associated and immunomodulatory proteins that are partially regulated by DNA methylation and copy number alterations. PD-1 expression and PD-L1 promoter methylation levels might be a prognostic marker for mucosal melanomas.

Mucosal melanomas of different anatomic sites share a common global DNA methylation profile with cutaneous melanoma but show location-dependent patterns of genetic and epigenetic alterations.

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non-cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location-dependent DNA methylation differences in cancer-related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location-dependent differences of promoter methylation frequencies in specific cancer-related genes together with tumor site-specific enrichment for specific chromosomal changes and genetic mutations. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

How the introduction of OSCEs has affected the time students spend studying: results of a nationwide study.

BACKGROUND: Medical schools globally now use objective structured clinical examinations (OSCEs) for assessing a student's clinical performance. In Germany, almost all of the 36 medical schools have incorporated at least one summative OSCE into their clinical curriculum. This nationwide study aimed to examine whether the introduction of OSCEs shifted studying time. The authors explored what resources were important for studying in preparation for OSCEs, how much time students spent studying, and how they performed; each compared to traditionally used multiple choice question (MCQ) tests. METHODS: The authors constructed a questionnaire comprising two identical sections, one for each assessment method. Either section contained a list of 12 study resources requesting preferences on a 5-point scale, and two open-ended questions about average studying time and average grades achieved. During springtime of 2015, medical schools in Germany were asked to administer the web-based questionnaire to their students in years 3-6. Statistical analysis compared the responses on the open-ended questions between the OSCE and MCQs using a paired t-test. RESULTS: The sample included 1131 students from 32 German medical schools. Physical examination courses were most important in preparation for OSCEs, followed by class notes/logs and the skills lab. Other activities in clinical settings (e.g. medical clerkships) and collaborative strategies ranked next. Conversely, resources for gathering knowledge (e.g. lectures or textbooks) were of minor importance when studying for OSCEs. Reported studying time was lower for OSCEs compared to MCQ tests. The reported average grade, however, was better on OSCEs. CONCLUSIONS: The study findings suggest that the introduction of OSCEs shifted studying time. When preparing for OSCEs students focus on the acquisition of clinical skills and need less studying time to achieve the expected level of competence/performance, as compared to the MCQ tests.

A pilot survey of student perceptions on the benefit of the OSCE and MCQ modalities.

Objective: The objective structured clinical examination (OSCE) has become widely accepted as a form of assessment in medical education. At the same time, the more traditional multiple choice question (MCQ) examinations remain a central modality of student assessment. This pilot survey aimed to investigate students' perceptions about the benefits of the OSCE and MCQs to yield data supporting the implementation of this assessment strategy into the national medical licensing examination in Germany. Methods: A questionnaire was delivered electronically to 34 German medical schools. Students in years 3-6 were invited to rate 11 items about objectives of good medical assessment. All items were presented for both the OSCE and MCQs using a 5-point Likert Scale (1=strongly disagree to 5=strongly agree). Factor analysis was used to identify underlying components in the ratings. Average scores of items that belonged to a component were computed. Results: Data analysis included 1,082 students from 32 medical schools. For the OSCE, factor analysis revealed two components, which were labelled "educational impact" and "development of clinical competence". The average scores of items were 3.37 and 3.55, respectively. For the MCQ modality, also two components emerged. These were labelled "perceived weaknesses of MCQs" and "perceived strengths of MCQs" (consisting of items such as "promotes my theoretical knowledge"). The average scores for these components were 1.85 and 3.62. Conclusion: The results of this pilot survey indicate that students consider both OSCE and MCQs as useful assessments for the purposes for which they were designed. The assessment strategy thus appears appropriate and it should be used in the national licensing examination.

Analysis of PIK3CA mutations in breast cancer subtypes.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) is a central element of a signaling pathway involved in cell proliferation, survival, and growth. Certain mutations in this pathway result in enhanced PI3K signaling, which is associated with oncogenic cellular transformation and cancer. The aims of this study were to characterize different types of PIK3CA mutations in exons 9 and 20 in a series of primary breast carcinomas and to correlate the results with clinicopathologic parameters and survival. We used frozen tissue samples and sequenced exons 9 and 20 for a series of 241 patients with a diagnosis of breast carcinoma. We found that 15.8% of the primary breast carcinomas possessed PIK3CA mutations in either exon 9 or exon 20. The rate of PIK3CA mutations was increased in HR(+)/HER2(-) tumors (18.6%), but this difference did not reach a statistical significance. The lowest rate of mutations was observed in HR(+)/HER2(+) tumors (5.3%). No statistically significant association was found between the presence of PIK3CA mutations and the prognostic/clinical features of breast cancer, including histologic subtype, Her2 status, axillary lymph node involvement, tumor grade, and tumor stage. However, the presence of the H1047R mutation in 10 samples was associated with a statistically significantly worse overall survival. PIK3CA mutation was found to be a frequent genetic change in all breast cancer subtypes but occurred with the highest rate in HR(+)/HER2(-) tumors. Further studies are needed to validate the prognostic impact of different PIK3CA mutations.

Transcription of the human EAP1 gene is regulated by upstream components of a puberty-controlling Tumor Suppressor Gene network.

Mammalian puberty is initiated by an increased pulsatile release of gonadotropin-releasing hormone (GnRH) from specialized neurons located in the hypothalamus. GnRH secretion is controlled by neuronal and glial networks, whose activity appears to be coordinated via transcriptional regulation. One of the transcription factors involved in this process is thought to be the recently described gene Enhanced at Puberty 1 (EAP1), which encodes a protein with dual transcriptional activity. In this study we used gene reporter and chromatin immunoprecipitation (ChIP) assays to examine the hypothesis that EAP1 expression is controlled by transcriptional regulators earlier postulated to serve as central nodes of a gene network involved in the neuroendocrine control of puberty. These regulators include Thyroid Transcription Factor 1 (TTF1), Yin Yang 1 (YY1), and CUX1, in addition to EAP1 itself. While TTF1 has been shown to facilitate the advent of puberty, YY1 (a zinc finger protein component of the Polycomb silencing complex) may play a repressive role. The precise role of CUX1 in this context is not known, but like EAP1, CUX1 can either activate or repress gene transcription. We observed that DNA segments of two different lengths (998 and 2744bp) derived from the 5'-flanking region of the human EAP1 gene display similar transcriptional activity. TTF1 stimulates transcription from both DNA segments with equal potency, whereas YY1, CUX1, and EAP1 itself, behave as transcriptional repressors. All four proteins are recruited in vivo to the EAP1 5'-flanking region. These observations suggest that EAP1 gene expression is under dual transcriptional regulation imposed by a trans-activator (TTF1) and two repressors (YY1 and CUX1) previously postulated to be upstream components of a puberty-controlling gene network. In addition, EAP1 itself appears to control its own expression via a negative auto-feedback loop mechanism. Further studies are needed to determine if the occupancy of the EAP1 promoter by these regulatory factors changes at the time of puberty.

Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer.

BACKGROUND: The human nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) mediates the nuclear export of proteins and messenger RNAs and, thus, is an important regulator of subcellular distribution of key molecules. Whereas cell-biologic studies have suggested a fundamental role for CRM1 in the regulation of mitosis, the expression of this protein in human tumor tissue has not been investigated to date. METHODS: In this study, the expression of CRM1 was analyzed in a cohort of 88 ovarian tumors and 12 ovarian cell lines for the first time to the authors' knowledge. RESULTS: Immunohistochemistry revealed increased nuclear (52.7%) and cytoplasmic (56.8%) expression of CRM1 in 74 carcinomas compared with the expression revealed in borderline tumors and benign lesions. Similarly, CRM1 expression was increased in ovarian cancer cell lines compared with human ovarian surface epithelial cells. Cytoplasmic CRM1 expression was related significantly to advanced tumor stage (P= .043), poorly differentiated carcinomas (P= .011), and higher mitotic rate (P= .008). Nuclear CRM1 was associated significantly with cyclooxygenase-2 (COX-2) expression (P= .002) and poor overall survival (P= .01). Because it was demonstrated previously that blocking of CRM1 by leptomycin B (LMB) contributes to the inhibition of nuclear export, the authors used a set of mechanistic assays to study the effects of CRM1 inhibition in cancer cells. Treatment of OVCAR-3 cells with LMB revealed a significant reduction of cell proliferation and increased apoptosis as well as suppressed interleukin-1beta-induced COX-2 expression. CONCLUSIONS: The current results indicated that CRM1 is expressed in a subpopulation of ovarian carcinomas with aggressive behavior and is related to poor patient outcome. A correlation also was demonstrated between CRM1 and COX-2 expression in ovarian cancer tissue. Furthermore, the treatment of ovarian cancer cells with LMB revealed a reduction in COX-2 expression. Therefore, the authors suggest that CRM1 may be an interesting biomarker for the assessment of patient prognosis and a molecular target for anticancer treatment.

Overexpression of cyclooxygenase-2 in human prostate carcinoma and prostatic intraepithelial neoplasia-association with increased expression of Polo-like kinase-1.

BACKGROUND: Cyclooxygenases (COX) as well as Polo-like kinases (PLK) are involved in proliferation and cell cycle regulation and have been suggested for preventive and therapeutic approaches in prostate carcinoma. METHODS: In this study, we studied expression and prognostic impact of COX-2 in invasive prostate carcinoma, prostatic intraepithelial neoplasia (PIN), atrophic glands, and normal prostatic glands, and investigated the association between COX-2 and PLK-1. RESULTS: We observed a positivity for COX-2 in 72.1% of PIN and in 44.7% of prostate carcinomas with an overexpression of COX-2 in prostate cancer and PIN compared to benign prostatic tissue (P < 0.0005). Furthermore, we observed a strong correlation between expression of PLK-1 and COX-2 (P < 0.0005). CONCLUSIONS: To our knowledge, this is the first report of a correlation between COX-2 and PLK-1 in a malignant tumor. COX-2 and PLK-1 may be interesting targets for new molecular therapies in prostate cancer.

Specific inhibition of AKT2 by RNA interference results in reduction of ovarian cancer cell proliferation: increased expression of AKT in advanced ovarian cancer.

The protein kinase AKT is involved in several signaling pathways that are important for tumor development and progression, suggesting that AKT might be an interesting target for a molecular tumor therapy. In this study, we investigated the AKT expression in ovarian carcinomas and the role of the AKT isoforms to ovarian cancer cell proliferation. We observed an increased AKT expression in 58% of the primary ovarian carcinomas as compared to normal ovaries by immunohistochemistry. AKT expression was significantly associated with positive lymph node status (P=0.002) and advanced FIGO stage (P=0.009). In western blot analysis, total AKT was expressed in all ovarian cancer cell lines and HOSE cells, while phosphorylated AKT was only observed in OVCAR-3 and SKOV-3 cells. The isoforms AKT1 and AKT2 were expressed at the mRNA level in all cell lines, while no relevant AKT3 mRNA levels were detected by conventional and quantitative RT-PCR. To determine the effects on cell proliferation, we used the unselective PI3K-inhibitor LY294002 as well as RNA interference to selectively inhibit the AKT isoforms. Treatment with LY294002 and the AKT2 siRNA reduced proliferation of OVCAR-3 cells. Our results show that AKT is expressed in a subpopulation of advanced ovarian carcinomas suggesting a role for this protein in the progression of this entity. Deactivation of AKT, especially AKT2 can result in reduction of cell growth. Accordingly, AKT is an interesting target for therapeutic intervention in ovarian cancer.

Expression of the ELAV-like protein HuR in human colon cancer: association with tumor stage and cyclooxygenase-2.

There is growing body of evidence that post-translational events contribute-in addition to genetic changes-to the progression of malignant tumors. These post-translational alterations may provide targets for new therapeutic approaches. The ELAV-like protein HuR stabilizes a group of cellular mRNAs which contain AU-rich elements in their 3' untranslated region. To investigate a possible contribution of post-translational changes to the progression of colon cancer and to overexpression of COX-2, we studied expression of HuR and COX-2 a cohort of colorectal adenocarcinomas and in colon cancer cell lines. All cell lines showed an expression of HuR mRNA and protein. In tumor tissue of colon carcinomas we observed two different staining patterns of HuR: A nuclear expression in 98% as well as an additional cytoplasmic expression in 53% of cases. COX-2 was expressed in 63% of carcinomas. Cytoplasmic expression of HuR was significantly associated with increased COX-2 expression as well as with high tumor stage. In univariate Kaplan-Meier analysis, grading, tumor stage and nodal status but not HuR or COX-2 expression were prognostic factors for overall survival. Our results suggest that the overexpression of HuR in colon cancer may be part of a regulatory pathway that controls the mRNA stability of cyclooxygenase-2 and provides an interesting example for a contribution of a dysregulation of mRNA stability to the progression of colorectal cancer. Based on our results, further studies are necessary to investigate whether HuR might be a potential target for a molecular tumor therapy.

Expression of estrogen receptor-related receptors, a subfamily of orphan nuclear receptors, as new tumor biomarkers in ovarian cancer cells.

A subfamily of orphan receptors, estrogen receptor-related receptors (ERRs), has been demonstrated to modulate the transcription of some estrogen responsive genes via variant estrogen response elements (EREs). This study was conducted to determine whether human ERRalpha, ERRbeta, and ERRgamma might be involved in the tumorigenesis of ovarian cancer. RT-PCR was performed to analyze the expression of hERRalpha, hERRbeta, hERRbeta-2, and hERRgamma mRNA in five ovarian cancer cell lines as well as 33 samples of ovarian cancer and 12 samples of normal ovary. Serum CA-125 levels were also analyzed in all samples by ELISA. Progression-free survival and overall survival of patients with different expression of ERRs were analyzed by the Kaplan-Meier method. To analyze the subcellular localization of ERRalpha, a green fluorescent protein (GFP)-reporter plasmid of hERRalpha was constructed and transfected into the ovarian cancer cell line OVCAR-3. Expression of hERRalpha-GFP fusion protein was observed in the nucleus of OVCAR-3 ovarian cancer cell lines. We observed increased expression of hERRalpha mRNA (P = 0.020) and hERRgamma mRNA (P = 0.045) in ovarian cancers compared to normal ovaries. In contrast, hERRbeta was only observed in 9.1% of ovarian cancers. We found a positive correlation between the serum CA-125 levels and hERRalpha expression (P = 0.012), but not hERRbeta and hERRgamma expression. Survival analysis showed that the hERRalpha-positive group has a reduced overall survival (P = 0.015), and the ERRgamma-positive group has a longer progression-free survival (P = 0.020). In multivariate analysis, expression of hERRalpha was an independent prognostic factor for poor survival (relative risk, 3.032; 95% CI, 1.27-6.06). Based on our results, ERRs may play an important role in ovarian cancer. hERRalpha may represent a biomarker of poor prognosis, and hERRgamma may be a new therapeutic target in ovarian cancer.

Alteration of homocysteine catabolism in pre-eclampsia, HELLP syndrome and placental insufficiency.

Hyperhomocysteinemia is a risk factor in obstetrical complications such as pre-eclampsia, 'hemolysis, elevated liver enzymes, low platelet' (HELLP)-syndrome and placental insufficiency. The aim of our study was to investigate the alterations of homocysteine catabolism in these patients in relation to serum B-vitamins and renal function. Maternal fasting serum from pre-eclampsia (n=24), HELLP (n=20) and placental insufficiency (n=25) patients at the time of diagnosis and pregnant controls (n=34) was analyzed for homocysteine and its metabolites cystathionine and methylmalonic acid, the vitamins B6, B12 and folate, renal and additional parameters. Cystathionine, a parameter of homocysteine catabolism, was significantly increased in pre-eclampsia and HELLP compared with controls and placental insufficiency patients (mean concentrations: 343, 324, 248, 227 nmol/l; p=0.001). Homocysteine, folic acid, vitamin B6 and methylmalonic acid, however, did not differ significantly between groups. The main determinants of cystathionine are cystatin C and vitamin B6, whereas the main determinants of homocysteine are folate and uric acid. The strongest dependency of cystathionine on vitamin B6 was observed in pre-eclampsia and HELLP patients. The results suggest that the vitamin B6-dependent trans-sulfuration pathway is activated in pre-eclampsia and HELLP syndrome, probably by oxidative stress. Therefore, the demand for vitamin B6 is increased in these patients. Furthermore, renal dysfunction and low vitamin B6 levels contribute to the increase of cystathionine in pre-eclampsia and HELLP patients.

Overexpression of the embryonic-lethal abnormal vision-like protein HuR in ovarian carcinoma is a prognostic factor and is associated with increased cyclooxygenase 2 expression.

The human embryonic-lethal abnormal vision-like protein HuR is involved in the regulation of mRNA turnover and serves as a shuttling protein between the nucleus and the cytoplasm that stabilizes mRNAs containing adenine- and uridine-rich elements in their 3' untranslated region. We have shown recently that expression of cyclooxygenase (COX)-2 is related to poor prognosis in ovarian carcinoma. Other studies have shown that the COX-2 mRNA contains an adenine- and uridine-rich element and is stabilized by HuR. In this study, we investigated the expression and cellular distribution of HuR in 83 primary ovarian carcinomas, 16 borderline tumors of the ovary, 3 normal ovaries, and 9 ovarian carcinoma cell lines. Expression of HuR was detected in all cell lines on the mRNA and protein level and showed a predominantly nuclear staining in OVCAR-3 cells by confocal microscopy. In an immunohistochemical evaluation of human ovarian carcinomas, HuR showed a nuclear expression in 81% of tumors. In addition, a cytoplasmic expression of HuR was observed in a subgroup of 45% of ovarian carcinomas. Nuclear as well as cytoplasmic expression of HuR was significantly increased in ovarian carcinomas compared with borderline tumors or normal ovaries. In univariate analysis, a significant association between cytoplasmic HuR expression and increased COX-2 expression (P = 0.025) as well as between histological grade (P = 0.008) and mitotic activity (P = 0.002) was observed, although nuclear expression of HuR was not correlated with COX-2 expression or other clinicopathological parameters. In Kaplan-Meier survival analysis, increased cytoplasmic expression of HuR was a significant prognostic indicator for progression-free survival (P = 0.03) as well as overall survival (P = 0.007). In multivariate analysis using the Cox regression model, cytoplasmic expression of HuR was an independent prognostic parameter for reduced overall survival with a relative risk of 2.62 (95% confidence interval, 1.32-5.19). Our results suggest that there is a dysregulation of cellular distribution of the mRNA stability factor HuR in a subset of invasive ovarian carcinomas. This dysregulation appears to result in an increased expression of COX-2, an increased proliferative rate, and may lead to a reduced survival time. Additional studies are required to analyze the downstream effects of increased cytoplasmic expression of HuR. In addition, it would be interesting to investigate the prognostic role of increased cytoplasmic expression of HuR in prospective studies.

Induction of G0/G1 cell cycle arrest in ovarian carcinoma cells by the anti-inflammatory drug NS-398, but not by COX-2-specific RNA interference.

Cyclooxygenases, particularly COX-2, play an important role in tumor development and progression. We have previously shown that COX-2 expression is an independent prognostic factor in human ovarian carcinoma. In this study, we investigated the effects of the inhibition of COX isoforms by the NSAID NS-398 as well as by COX-isoform-specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1 and an induction of high levels of COX-2 and PGE(2) after stimulation with interleukin-1beta. In contrast, SKOV-3 cells were negative for both COX isoforms. In OVCAR-3 cells, PGE(2) production was inhibited by NS-398 in concentrations of 1 microM and by a COX-2-specific silencing RNA (siRNA), while a COX-1-specific siRNA did not have an effect. This suggests that COX-2 is the major source of PGE(2) in this cell line. To dissociate COX-2-specific and non-COX-2-specific effects on cell proliferation, a proliferation assay was performed after incubation of cells with NS-398 and COX siRNAs. NS-398 induced an inhibition of cell proliferation at concentrations of 50-500 microM, which are above the concentrations needed for the inhibition of PGE(2) production. This inhibitory effect was present in the COX-positive cell line OVCAR-3 as well as in the COX-negative cell line SKOV-3 and could not be reverted by addition of exogenous PGE(2). Neither COX-1- nor COX-2-specific siRNAs had an effect on cell proliferation of OVCAR-3 cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with NS-398, but not with COX siRNAs. These experiments suggest that NS-398 reduced cell proliferation in ovarian carcinoma cells by induction of G0/G1 cell cycle arrest independent of COX-2 inhibition. Our study shows that specific inhibition of COX isoforms by RNAi could be used to dissociate effects of NSAIDs. Furthermore, our results suggest that cell cycle arrest is one of the primary mechanisms responsible for the antiproliferative effects of NS-398 on ovarian carcinoma cells.

Cytokine-suppressive anti-inflammatory drugs (CSAIDs) inhibit invasion and MMP-1 production of ovarian carcinoma cells.

The development of therapeutic strategies for inhibition of peritoneal dissemination and invasion would be central for the treatment of ovarian carcinoma. In the microenvironment of ovarian carcinomas, various inflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) are present. In this study we investigated the role of inflammatory cytokines in the regulation of invasion of SKOV-3 ovarian carcinoma cells in-vitro. Treatment of cells with TNF-alpha or interleukin 1beta (IL-1beta) lead to increased phosphorylation of the stress-activated p38 mitogen-activated protein kinase (p38MAPK). Furthermore, TNF-alpha as well as IL-1beta stimulated matrigel invasion of tumor cells. An inhibitor of stress-activated protein kinase pathways, the cytokine-suppressive anti-inflammatory drug (CSAID) SB203580 inhibited invasion of cytokine-stimulated SKOV-3 cells. The MEK-1 inhibitor PD98059 similarly inhibited invasion of cytokine-stimulated cells, but to a lesser extent. Expression of mRNA and protein levels of matrix metalloproteinase-1 (MMP-1) by SKOV-3 cells could be stimulated by inflammatory cytokines and inhibited by SB203580, and partially also by PD98059. Our results show that CSAIDs reduce invasion and MMP expression of ovarian carcinoma cells. Further studies are required to investigate whether inhibition of cytokine-induced signal transduction may be of value in therapy of ovarian carcinomas in-vivo.

Genetic alterations in epithelial ovarian tumors analyzed by comparative genomic hybridization.

The genetic changes involved in the pathogenesis of ovarian carcinoma are not completely understood. To investigate this matter, we studied paraffin-embedded, microdissected tissue of 47 ovarian epithelial tumors (9 adenomas, 11 tumors of low malignant potential [LMP], 14 serous carcinomas, and 13 nonserous carcinomas) using comparative genomic hybridization (CGH). (The primary data used in this study are available at our CGH online tumor database at http://amba.charite.de/cgh.) Chromosomal imbalances were found in 1 serous adenoma and in 7 LMP tumors. In the latter the alterations appeared randomly and showed no overlap with alterations found in invasive carcinomas. Although the mean aberration number of low-grade serous carcinomas was comparable to LMP tumors, the imbalances of the former occurred with high incidence (>50%) and were found at different localizations. High-grade serous carcinomas had more than twice as much chromosomal imbalances as low-grade serous carcinomas and also had pronounced alterations. In serous carcinomas, gains were found on 3q, 6p, 7, 8q, and 20, and losses were found on 4q, 6q, 12q, 13q, and 16q. Comparing serous and nonserous carcinomas, the mean aberration number was comparable, but the number of high incidence changes was lower, and the most frequent imbalances were losses on 13q and gains on 20p. Overlapping alterations occurring in serous and nonserous carcinomas were gains on 3q and 6p, as well as losses on 4q. Chromosomal imbalances associated with poor prognosis of ovarian carcinomas were gains on 6p, 7q, and 13q and losses on 15q, 17p, 18q, and 21q. Our data indicate that serous LMP tumors and invasive carcinomas have different genetic aberrations, indicating that invasive carcinomas do not arise from preexisting serous LMP tumors. On the other hand, there are common genetic abnormalities in serous and nonserous carcinomas, suggesting that they have very early lesions in common but take different paths of further development.

Expression of cyclooxygenase 2 is an independent prognostic factor in human ovarian carcinoma.

Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme in prostanoid biosynthesis and is involved in tumor progression. We investigated expression of COX-1 and COX-2 in cell lines and tumors from ovarian carcinomas. Expression of COX-2 mRNA and protein was detectable in three of five ovarian carcinoma cell lines and was inducible by interleukin-1beta or phorbolester in a subset of cell lines. Prostaglandin E(2) (PGE(2)) production could be inhibited by the selective COX-2 inhibitor NS-398. In malignant ascites of ovarian carcinomas significantly increased levels of PGE(2) were found compared to other carcinomas or nonmalignant ascites (P = 0.03). We investigated expression of COX-2 by immunohistochemistry in 117 ovarian surface epithelial tumors. Expression of COX-2 was detected in 42% of 86 ovarian carcinomas and in 37% of 19 low malignant potential tumors, but not in 12 cystadenomas or 2 normal ovaries. Expression of COX-1 was detected by immunohistochemistry in 75% of 75 invasive ovarian carcinomas and in 75% of 16 low malignant potential tumors, whereas 2 samples from normal ovaries and 8 cystadenomas were positive for COX-1. In univariate survival analysis of invasive carcinomas, expression of COX-2 was associated with a significantly reduced median survival time (log rank test, P = 0.04). For patients younger than 60 years of age, this association was even more significant (P < 0.004). In contrast, expression of COX-1 was no prognostic parameter (P = 0.89). There was no significant correlation between COX-2 or COX-1 expression and other clinicopathological markers. In multivariate analysis expression of COX-2 was an independent prognostic factor for poor survival (relative risk, 2.74; 95% CI, 1.38 to 5.47). Our data indicate that COX-2 expression is an independent prognostic factor in ovarian carcinoma. Based on the results of this study, it would be interesting to investigate whether ovarian carcinoma patients with tumors positive for COX-2 would benefit from treatment with selective COX-2 inhibitors.