Jayne L. Dennis

Markers of Adenocarcinoma Characteristic of the Site of Origin: Development of a Diagnostic Algorithm

Purpose: Patients with metastatic adenocarcinoma of unknown origin are a common clinical problem. Knowledge of the primary site is important for their management, but histologically, such tumors appear similar. Better diagnostic markers are needed to enable the assignment of metastases to likely sites of origin on pathologic samples. Experimental Design: Expression profiling of 27 candidate markers was done using tissue microarrays and immunohistochemistry. In the first (training) round, we studied 352 primary adenocarcinomas, from seven main sites (breast, colon, lung, ovary, pancreas, prostate and stomach) and their differential diagnoses. Data were analyzed in Microsoft Access and the Rosetta system, and used to develop a classification scheme. In the second (validation) round, we studied 100 primary adenocarcinomas and 30 paired metastases. Results: In the first round, we generated expression profiles for all 27 candidate markers in each of the seven main primary sites. Data analysis led to a simplified diagnostic panel and decision tree containing 10 markers only: CA125, CDX2, cytokeratins 7 and 20, estrogen receptor, gross cystic disease fluid protein 15, lysozyme, mesothelin, prostate-specific antigen, and thyroid transcription factor 1. Applying the panel and tree to the original data provided correct classification in 88%. The 10 markers and diagnostic algorithm were then tested in a second, independent, set of primary and metastatic tumors and again 88% were correctly classified. Conclusions: This classification scheme should enable better prediction on biopsy material of the primary site in patients with metastatic adenocarcinoma of unknown origin, leading to improved management and therapy.

In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts

Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.

Behavioural responses of Mallorcan midwife toad tadpoles to natural and unnatural snake predators

The activity levels of Mallorcan midwife toad tadpoles, Alytes muletensis, were compared in two natural torrent pools which differed in their use by predatory viperine snakes, Natrix maura. Activity levels were lower in a pool regularly used by snakes than they were in a snake-free pool, but were reduced in both pools when snakes were experimentally introduced in nylon bags. In the presence of snakes, however, activity was more suppressed in the pool that was usually snake-free. Corresponding reductions in activity were also observed when tadpoles were treated with chemical cues from Mallorcan N. maura in a gravitational flow-through system. However, tadpoles failed to respond to chemical cues from other species of amphibian-eating snakes, or even to those from N. maura collected from a different population in mainland Spain. As none of the snakes used had previously eaten midwife toads, the responses cannot be related to previous diet, and seem to be specific to those N. maura from the island of Mallorca. As viperine snakes were probably introduced to Mallorca about 2000 years ago, the evolution of anti-predator behaviour in midwife toad tadpoles must have occurred relatively recently. Copyright 1998 The Association for the Study of Animal Behaviour.

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

BackgroundEndothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown.ResultsUsing microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated.ConclusionThe phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response.

Hunting the primary: novel strategies for defining the origin of tumours

In 1995, two methods of genome‐wide expression profiling were first described: expression microarrays and serial analysis of gene expression (SAGE). In the subsequent 10 years, many hundreds of papers have been published describing the application of these technologies to a wide spectrum of biological and clinical questions. Common to all of this research is a basic process of data gathering and analysis. The techniques and statistical and bio‐informatic tools involved in this process are reviewed. The processes of class discovery (using clustering and self‐organizing maps), class prediction (weighted voting, k nearest neighbour, support vector machines, and artificial neural networks), target identification (fold change, discriminant analysis, and principal component analysis), and target validation (RT‐PCR and tissue microarrays) are described. Finally, the diagnostic problem of adenocarcinomas that present as metastases of unknown origin is reviewed, and it is demonstrated how integration of expression profiling techniques promises to throw new light on this important clinical challenge. Copyright

MiMiR – an integrated platform for microarray data sharing, mining and analysis

BackgroundDespite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Mi croarray data Mi ning R esource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data.ResultsA user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package.ConclusionThe new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centres large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies.

Myocardial depressant effects of interleukin 6 in meningococcal sepsis are regulated by p38 mitogen-activated protein kinase.

Objectives:Myocardial failure, leading to inotrope-unresponsive shock, is the predominant cause of death in meningococcal and other forms of septic shock. Proinflammatory cytokines released in septic shock are known to have myocardial depressant effects. We previously showed that interleukin 6 is a major myocardial depressant factor in children with meningococcal septicemia. In the current study, we aimed to investigate the mechanisms by which interleukin 6 induces myocardial failure in meningococcal sepsis and to identify potential novel therapeutic targets. Design:Laboratory-based study. Setting:University hospital and laboratories. Patients:Children with a clinical diagnosis of meningococcal septic shock. Methods:We studied interleukin 6-induced signaling events, both in vitro using isolated rat ventricular cardiac myocytes as a model of myocardial contractility and in whole blood from children with meningococcal sepsis. Interventions:None. Measurements and Main Results:We demonstrated involvement of Janus kinase 2, phosphatidylinositol 3-kinase, Akt, and p38 mitogen-activated protein kinase in interleukin 6-induced negative inotropy in isolated cardiac myocytes. Inhibition of p38 mitogen-activated protein kinase not only reversed interleukin 6-induced myocardial depression in both rat and human myocytes, but restored inotrope responsiveness. Cardiomyocytes transduced with dominant-negative p38 mitogen-activated protein kinase showed no interleukin 6-induced myocardial depression. To investigate p38 mitogen-activated protein kinase in vivo, we profiled global RNA expression patterns in peripheral blood of children with meningococcal septicemia. Transcripts for genes mapping to the p38 mitogen-activated protein kinase pathway showed significantly altered levels of abundance with a high proportion of genes of this pathway affected. Conclusions:Our findings demonstrate an integral role of the p38 mitogen-activated protein kinase pathway in interleukin 6-mediated cardiac contractile dysfunction and inotrope insensitivity. Dysregulation of the p38 mitogen-activated protein kinase pathway in meningococcal septicemia suggests that this pathway may be an important target for novel therapies to reverse myocardial dysfunction in patients with meningococcal septic shock who are not responsive to inotropic support.

The gene expression profile of unstimulated dendritic cells can be used as a predictor of function.

Dendritic cells (DCs) represent a subset of professional antigen presenting cell (APC) whose role is to elicit immune responses against harmful antigens. They have been used in DC vaccines to stimulate the immune system to kill cancer cells. However, successes in clinical trials have been limited, which may be attributed to a lack of appreciation of the quality of DCs used. In the present study, whole human genome microarrays were used to examine alterations in gene expression of monocyte‐derived DCs after stimulation with supernatants derived from tumours. Our primary aim was to investigate the possibility of a gene signature for DCs that could be used to forecast responsiveness to tumour stimuli. Results showed that DCs are divided into two groups based on their ability to increase costimulatory markers and to trigger T‐cell responses. The gene profiles of the immature DCs from these two groups were distinct, with particular divergence in genes from the interleukin (IL) 8 and thrombospondin‐1 hubs. A subpanel of genes was identified, whose signature of expression was capable of predicting DC‐stimulatory capacity. Overall, these studies have highlighted a gene‐based screen that predicts DC function, which could be used to guide DC‐vaccine trials.

Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis

Background: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. Methods: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. Results: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. Conclusion: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

The type and quality of paraffin wax is important when constructing tissue microarrays

Tissue microarray (TMA) technology allows the representation of hundreds of tissue samples on a standard microscope slide. This is achieved by arraying small cores (0.6 mm in diameter) of paraffin wax embedded tissue samples in a recipient wax block. Sections cut from the array can then be assessed by immunohistochemistry or in situ hybridisation, according to standard protocols. TMAs enable the high throughput assessment of the presence and location of expressed genes, saving time, reagents, and clinical material. There have been several reviews on TMA construction and use,1 but we have recently encountered a technical problem that, as far as we are aware, has not been described in the literature. Multiple TMAs, each containing 292 cores, were constructed according to standard protocols.2 Sections were cut without mishap using the adhesive …