Katherine M. Sheehan

Use of Reverse Phase Protein Microarrays and Reference Standard Development for Molecular Network Analysis of Metastatic Ovarian Carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient’s metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer.

The human proteome, due to the enormity of post‐translational permutations that result in large numbers of isoforms, is much more complex than the genome and alterations in cancer can occur in ways that are not predictable by translational analysis alone. Proteomic analysis therefore represents a more direct way of investigating disease at the individual patient level. Furthermore, since most novel therapeutic targets are proteins, proteomic analysis potentially has a central role in patient care. At the same time, it is becoming clear that mapping entire networks rather than individual markers may be necessary for robust diagnostics as well as tailoring of therapy. Consequently, there is a need for high‐throughput multiplexed proteomic techniques, with the capability of scanning multiple cases and analysing large numbers of endpoints. New types of protein arrays combined with advanced bioinformatics are currently being used to identify molecular signatures of individual tumours based on protein pathways and signalling cascades. It is envisaged that analysing the cellular ‘circuitry’ of ongoing molecular networks will become a powerful clinical tool in patient management. Copyright

Dimensions of perceived control: A factor analysis of three measures and an examination of their relation to activity level and mood in a student and cross-cultural patient sample

Abstract This study investigated whether measures of three perceived control (PC) concepts tapped independent dimensions, were invariant to population characteristics of health and culture, were differentially sensitive to health outcomes, and were discriminable from self-report disability. The Multidimensional Health Locus of Control, Generalized Self-efficacy, and Perceived Health Competence (PHC) Scales were administered to 106 Scottish students and 145 Scottish, Spanish and Irish patients, who also completed versions of the Sickness Impact Profile. Principal Components analyses revealed five factors reflecting scale constructions for students and six factors for patients, as PHC items fell on two factors. Disability items fell on a separate factor from all PC items. Results suggest: (a) PC is multidimensional regardless of health and culture; (b) adequate assessment requires multiple PC measures; and (c) it is meaningful to consider PC as a possible influence on disability.

Protective Role for Caspase-11 during Acute Experimental Murine Colitis

Activation of the noncanonical inflammasome, mediated by caspase-11, serves as an additional pathway for the production of the proinflammatory cytokines IL-1β and IL-18. Noncanonical inflammasome activity occurs during host defense against Gram-negative bacteria and in models of acute septic shock. We propose that the noncanonical inflammasome is activated in mice during acute intestinal inflammation elicited by dextran sodium sulfate (DSS), a model of experimental colitis. We find that caspase-11−/− mice display enhanced susceptibility to DSS, because of impaired IL-18 production. The impaired IL-18 levels observed are shown to result in reduced intestinal epithelial cell proliferation and increased cell death. We also suggest that a novel type II IFN–dependent, type I IFN-TRIF–independent signaling pathway is required for in vivo caspase-11 production in intestinal epithelial cells during DSS colitis. Collectively, these data suggest that IFN-γ–mediated caspase-11 expression has a key role maintaining intestinal epithelial barrier integrity in vivo during experimentally induced acute colitis.

The relationship between cyclooxygenase-2 expression and characteristics of malignant transformation in human colorectal adenomas

Background and aims Cyclooxygenase 2 (COX-2) is a target of aspirin and other non-steroidal anti-inflammatory drugs and is implicated in the pathogenesis of colorectal cancer. The objective of this study was to evaluate the extent of COX-2 in pre-malignant colorectal polyps and to assess the relationship between COX-2 and the level of dysplasia in these lesions. Methods Whole polypectomy specimens were retrieved from 123 patients by endoscopic or surgical resection. Following formalin fixation and paraffin embedding, the polyps were evaluated histologically for size, type and grade of dysplasia. The extent of COX-2 expression was measured by the avidin–biotin immunohistochemical technique using a monoclonal COX-2 antibody. The extent of COX-2 expression was graded according to percentage epithelial COX-2 expression. Results The polyps were of the following histological types: 10 hyperplastic, 35 tubular adenomas, 61 tubulovillous adenomas and 17 villous adenomas. Twenty showed mild dysplasia, 65 moderate dysplasia, and 28 focal or severe dysplasia (including eight with focal invasion). The average polyp size was 1.7 cm. Nine hyperplastic polyps were COX-2-negative and one was COX-2-positive. COX-2 expression was more extensive in larger polyps and in polyps with a higher villous component. There was a significant increase in the extent of COX-2 protein with increasing severity of dysplasia. Within a polyp, there was a focal corresponding increase in COX-2 expression within epithelium showing a higher grade of dysplasia. Conclusions COX-2 expression is related directly to colorectal adenomatous polyp size, type and grade of dysplasia. This suggests that the role of COX-2 in colorectal cancer may be at an early stage in the adenoma-to-carcinoma sequence and supports the suggestion that inhibition of COX-2 may be useful chemoprevention for this disease.

GLUT‐1 expression and response to chemoradiotherapy in rectal cancer

Preoperative chemoradiotherapy is used in locally advanced rectal cancer to reduce local recurrence and improve operability, however a proportion of tumors do not undergo significant regression. Identification of predictive markers of response to chemoradiotherapy would improve patient selection and may allow response modification by targeting of specific pathways. The aim of this study was to determine whether expression of glucose transporter‐1 (GLUT‐1) and p53 in pretreatment rectal cancer biopsies was predictive of tumor response to chemoradiotherapy. Immunohistochemical staining for GLUT‐1 and p53 was performed on 69 pretreatment biopsies and compared to tumor response in the resected specimen as determined by the tumor regression grade (TRG) scoring system. GLUT‐1 expression was significantly associated with reduced response to chemoradiotherapy and increasing GLUT expression correlated with poorer response (p = 0.02). GLUT‐1 negative tumors had a 70% probability of good response (TRG3/4) compared to a 31% probability of good response in GLUT‐1 positive tumors. GLUT‐1 may be a useful predictive marker of response to chemoradiotherapy in rectal cancer.

The {Delta}Np63 Proteins Are Key Allies of BRCA1 in the Prevention of Basal-Like Breast Cancer.

Little is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless, we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells, siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability, increased cell proliferation, loss of DNA damage checkpoint control, and impaired growth control. Together, our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer.

Nonsteroidal anti-inflammatory drug-induced diaphragms and ulceration in the colon.

The toxic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the small bowel have been reported extensively. A growing number of reports of toxic effects of NSAIDs on the colon have appeared recently. The clinical presentation, endoscopic appearances and histological findings of so-called NSAID colopathy are quite varied, as illustrated by a series of four patients described in this report. Presenting symptoms and signs in this series include iron-deficiency anaemia and crampy abdominal pain, but alteration of bowel habit, weight loss, and even nausea and vomiting have also been described. One patient in this series has large-bowel diaphragms, considered by some to be pathognomonic of NSAID effects. Each of the four patients had right-sided colonic lesions only, possibly supporting a direct toxic effect of NSAIDs. Management usually involves simply stopping the offending NSAID. A review of the literature on this under-recognized entity is presented.

The prognostic value of tumour regression grade following neoadjuvant chemoradiation therapy for rectal cancer

To date, there is no uniform consensus on whether tumour regression grade (TRG) is predictive of outcome in rectal cancer. Furthermore, the lack of standardization of TRG grading is a major source of variability in published studies. The aim of this study was to evaluate the prognostic impact of TRG in a cohort of patients with locally advanced rectal cancer treated with neoadjuvant chemoradiation therapy (CRT). In addition to the Mandard TRG, we utilized four TRG systems modified from the Mandard TRG system and applied them to the cohort to assess which TRG system is most informative.

Stimulation of adhesion molecule expression by Helicobacter pylori and increased neutrophil adhesion to human umbilical vein endothelial cells

Helicobacter pylori upregulates endothelial adhesion molecules but the pattern is unclear. Human umbilical vein endothelial cells (HUVEC) were exposed to control medium or H. pylori 60190. Binding of monoclonal antibodies against P‐selectin, E‐selectin, vascular adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) was determined using enzyme‐linked immunosorbent assay. Binding of polymorphonuclear leukocytes to HUVEC was determined on cells exposed as above. After 6 h exposure to H. pylori, there were 30%, 124%, 167% and 100% increases in P‐selectin, E‐selectin, VCAM‐1 and ICAM‐1 levels and a 400% increase in polymorphonuclear leukocyte adhesion in HUVEC exposed to H. pylori. Effects of incubation for other intervals between 0 and 18 h are also described. H. pylori exerts some of its effects on gastric mucosa via gastric vasculature. This study gives insight into the pattern of H. pylori‐associated endothelial adhesion molecule upregulation.