Molecular pathology - The value of an integrative approach

Molecular Pathology (MP) is at the heart of modern diagnostics and translational research, but the controversy on how MP is best developed has not abated. The lack of a proper model or trained pathologists to support the diagnostic and research missions makes MP a rare commodity overall. Here we analyse the scientific and technology areas, in research and diagnostics, which are encompassed by MP of solid tumours; we highlight the broad overlap of technologies and analytical capabilities in tissue research and diagnostics; and we describe an integrated model that rationalizes technical know-how and pathology talent for both. The model is based on a single, accredited laboratory providing a single standard of high-quality for biomarker discovery, biomarker validation and molecular diagnostics.

Digital pathology and image analysis in tissue biomarker research

Digital pathology and the adoption of image analysis have grown rapidly in the last few years. This is largely due to the implementation of whole slide scanning, advances in software and computer processing capacity and the increasing importance of tissue-based research for biomarker discovery and stratified medicine. This review sets out the key application areas for digital pathology and image analysis, with a particular focus on research and biomarker discovery. A variety of image analysis applications are reviewed including nuclear morphometry and tissue architecture analysis, but with emphasis on immunohistochemistry and fluorescence analysis of tissue biomarkers. Digital pathology and image analysis have important roles across the drug/companion diagnostic development pipeline including biobanking, molecular pathology, tissue microarray analysis, molecular profiling of tissue and these important developments are reviewed. Underpinning all of these important developments is the need for high quality tissue samples and the impact of pre-analytical variables on tissue research is discussed. This requirement is combined with practical advice on setting up and running a digital pathology laboratory. Finally, we discuss the need to integrate digital image analysis data with epidemiological, clinical and genomic data in order to fully understand the relationship between genotype and phenotype and to drive discovery and the delivery of personalized medicine.

'O daughter ... forget your people and your father's house': Early Modern Women Writers and the Spanish Imaginary

‘O daughter … forget your people and your father’s house’: Early Modern Women Writers and the Spanish ImaginaryAnne Holloway and Ramona WrayHolloway and Wray consider the perspectives offered by two very different seventeenth-century women (Mary Bonaventure Browne, or Mother Browne (b.1615- and Lady Ann Fanshawe (b.1625) both of whom exchanged Ireland for Spain, and both of whom record journeys both ‘real’ and imagined in their writings. Browne’s deployment of hagiographical tropes in her History of the Poor Clares may reveal the potential impact of Iberian conventual culture; her allusions to the markers of sanctity insistent on the immutability of the body, whilst accepting and anticipating spectral presence in the form of bilocation. Fanshawe’s Memoirs are considered alongside the material legacy of her ‘Booke of Receipts of Physickes, Salues, Waters, Cordialls, Preserues and Cookery.’ Her impressions both in transit and within the domus are similarly marked by receptivity and sensitivity to the host culture. Amidst a backdrop of religious persecution and political uncertainty, in both cases Spain emerges as a potentially enabling context for creativity and self-expression.Keywords: Memoir; Franciscan; Poor Clares; Fanshawe; Mary Bonaventure Browne; hagiography; life-writing; autobiography, women writers

Secular trends in reported portion size of food and beverages consumed by Irish adults

The present analysis aimed to investigate the changes in the reported portion sizes (PS) of foods and beverages commonly consumed by Irish adults (18-64 years) from the North South Ireland Food Consumption Survey (NSIFCS) (1997-2001) and the National Adult Nutrition Survey (NANS) (2008-10). Food PS, which are defined as the weight of food (g) consumed per eating occasion, were calculated for comparable foods and beverages in two nationally representative cross-sectional Irish food consumption surveys and were published in NSIFCS and NANS. Repeated measure mixed model analysis compared reported food PS at the total population level as well as subdivided by sex, age, BMI and social class. A total of thirteen commonly consumed foods were examined. The analysis demonstrated that PS significantly increased for five foods ('white sliced bread', 'brown/wholemeal breads', 'all meat, cooked', 'poultry, roasted' and 'milk'), significantly decreased for three ('potatoes', 'chips/wedges' and 'ham, sliced') and did not significantly change for five foods ('processed potato products', 'bacon/ham', 'cheese', 'yogurt' and 'butter/spreads') between the NSIFCS and the NANS. The present study demonstrates that there was considerable variation in the trends in reported food PS over this period.

Business Intelligence and Multi-market Competition

We consider a multi-market framework where a set of firms compete on two oligopolistic markets. The cost of production of each firm allows for spillovers across markets, ensuring that output decisions for both markets have to be made jointly. Prior to competing in these markets, firms can establish links gathering business intelligence about other firms. A link formed by a firm generates two types of externalities for competitors and consumers. We characterize the business intelligence equilibrium networks and networks that maximize social welfare. By contrast with single market competition, we show that in multi-market competition there exist situations where intelligence gathering activities are underdeveloped with regard to social welfare and should be tolerated, if not encouraged, by public authorities.

PICan: An integromics framework for dynamic cancer biomarker discovery

Modern cancer research on prognostic and predictive biomarkers demands the integration of established and emerging high-throughput technologies. However, these data are meaningless unless carefully integrated with patient clinical outcome and epidemiological information. Integrated datasets hold the key to discovering new biomarkers and therapeutic targets in cancer. We have developed a novel approach and set of methods for integrating and interrogating phenomic, genomic and clinical data sets to facilitate cancer biomarker discovery and patient stratification. Applied to a known paradigm, the biological and clinical relevance of TP53, PICan was able to recapitulate the known biomarker status and prognostic significance at a DNA, RNA and protein levels.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor β-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modelled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.

Potentially harmful excipients in neonatal medicines: A pan-European observational study

Objectives We aimed to describe administration of eight potentially harmful excipients of interest (EOI)-parabens, polysorbate 80, propylene glycol, benzoates, saccharin sodium, sorbitol, ethanol and benzalkonium chloride-to hospitalised neonates in Europe and to identify risk factors for exposure. Methods All medicines administered to neonates during 1 day with individual prescription and demographic data were registered in a web-based point prevalence study. Excipients were identified from the Summaries of Product Characteristics. Determinants of EOI administration (geographical region, gestational age (GA), active pharmaceutical ingredient, unit level and hospital teaching status) were identified using multivariable logistical regression analysis. Results Overall 89 neonatal units from 21 countries participated. Altogether 2095 prescriptions for 530 products administered to 726 neonates were recorded. EOI were found in 638 (31%) prescriptions and were administered to 456 (63%) neonates through a relatively small number of products (n=142; 27%). Parabens, found in 71 (13%) products administered to 313 (43%) neonates, were used most frequently. EOI administration varied by geographical region, GA and route of administration. Geographical region remained a significant determinant of the use of parabens, polysorbate 80, propylene glycol and saccharin sodium after adjustment for the potential covariates including anatomical therapeutic chemical class of the active ingredient. Conclusions European neonates receive a number of potentially harmful pharmaceutical excipients. Regional differences in EOI administration suggest that EOI-free products are available and provide the potential for substitution to avoid side effects of some excipients.

PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

Although recent decades have seen an improved cure rate for newly diagnosed paediatric acute lymphoplastic leukaemia (ALL), the treatment options for adult ALL, T-cell ALL (T-ALL) and relapsed disease remain poor. We have developed a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds and established their anticancer efficacy in a variety of human tumour cell types. Here, we demonstrate that PBOX-15 inhibits cell growth, and induces G2/M cell cycle arrest and apoptosis in both T-ALL and B-cell ALL (B-ALL) cells. In addition, prior to PBOX-15-induced apoptosis, PBOX-15 decreases ALL cell adhesion, spreading and migration. Concurrently, PBOX-15 differentially down-regulates β1-, β2- and α4-integrin expression in ALL cells and significantly decreases integrin-mediated cell attachment. PBOX-15 interferes with the lateral mobility and clustering of integrins in both B-ALL and T-ALL cells. These data suggest that PBOX-15 is not only effective in inducing apoptosis in ALL cells, but also has the potential to disrupt integrin-mediated adhesion of malignant lymphocytes, which represents a novel avenue for regulating leukaemic cell homing and migration.

Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches

In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ-PCR) and 3-colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time-points during treatment (93.38%), and a combined use of both approaches allowed a multi time-point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0.01% were identified by RQ-PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ-PCR and FC approach, the evaluation of 367 follow-up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0.04), >0.01% at the end of induction (P = 0.02), >0.01% at the end of consolidation (P = 0.01), >0.01% prior to the first delayed intensification (P = 0.01), and >0.1% prior to the second delayed intensification and continued maintenance (P = 0.001) were all associated with relapse and, based on early time-points (end of induction and consolidation) a significant log-rank trend (P = 0.0091) was noted between survival curves for patients stratified into high, intermediate and low-risk MRD groups.