Herceptest: HER2 expression and gene amplification in non-small cell lung cancer

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3 + cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting. © 2001 Wiley-Liss, Inc.

Communication Interception Technology (CIT) and its use in the fight against Transnational Organised Crime (TOC) in Australia : a review of the literature

In recent times, technology has advanced in such a manner that the world can now communicate in means previously never thought possible. Transnational organised crime groups, who have exploited these new technologies as basis for their criminal success, however, have not overlooked this development, growth and globalisation. Law enforcement agencies have been confronted with an unremitting challenge as they endeavour to intercept, monitor and analyse these communications as a means of disrupting the activities of criminal enterprises. The challenge lies in the ability to recognise and change tactics to match an increasingly sophisticated adversary. The use of communication interception technology, such as phone taps or email interception, is a tactic that when used appropriately has the potential to cause serious disruption to criminal enterprises. Despite the research that exists on CIT and TOC, these two bodies of knowledge rarely intersect. This paper builds on current literature, drawing them together to provide a clearer picture of the use of CIT in an enforcement and intelligence capacity. It provides a review of the literature pertaining to TOC, the structure of criminal enterprises and the vulnerability of communication used by these crime groups. Identifying the current contemporary models of policing it reviews intelligence-led policing as the emerging framework for modern policing. Finally, it assesses the literature concerning CIT, its uses within Australia and the limitations and arguments that exist. In doing so, this paper provides practitioners with a clearer picture of the use, barriers and benefits of using CIT in the fight against TOC. It helps to bridge the current gaps in modern policing theory and offers a perspective that can help drive future research.

Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis

Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.

The role of the molecular footprint of EGFR in tailoring treatment decisions in NSCLC

The majority of patients with non-small-cell lung cancer (NSCLC) present with advanced disease, with targeted therapies providing some improvement in clinical outcomes. The epidermal growth factor receptor (EGFR) tyrosine kinase (TK) plays an important role in the pathogenesis of NSCLC. Tyrosine kinase inhibitors (TKIs), which target the EGFR TK domain, have proven to be an effective treatment strategy; however, patient responses to treatment vary considerably. Therefore, the identification of patients most likely to respond to treatment is essential to optimise the benefit of TKIs. Tumour-associated activating mutations in EGFR can identify patients with NSCLC who are likely to have a good response to TKIs. Nonetheless, the majority of patients relapse within a year of starting treatment. Studies of tumours at relapse have demonstrated expression of a T790M mutation in exon 20 of the EGFR TK domain in approximately 50% of cases. Although conferring resistance to reversible TKIs, these patients may remain sensitive to new-generation irreversible/panerb inhibitors. A number of techniques have been employed for genotypic assessment of tumourassociated DNA to identify EGFR mutations, each of which has advantages and disadvantages. This review presents an overview of the current methodologies used to identify such molecular markers. Recent developments in technology may make the monitoring of changes in patients' tumour genotypes easier in clinical practice, which may enable patients' treatment regimens to be tailored during the course of their disease, potentially leading to improved patient outcomes.

An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers

Background: To directly assess tumor oxygenation in resectable non - small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes. Methods: Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes. Results: The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO 2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001). Conclusions: Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors. © 2006 American Association for Cancer Research.

Gene expression analysis of diagnostic biopsies predicts pathological response to neoadjuvant chemoradiotherapy of esophageal cancer

OBJECTIVE: This study explored gene expression differences in predicting response to chemoradiotherapy in esophageal cancer. PURPOSE:: A major pathological response to neoadjuvant chemoradiation is observed in about 40% of esophageal cancer patients and is associated with favorable outcomes. However, patients with tumors of similar histology, differentiation, and stage can have vastly different responses to the same neoadjuvant therapy. This dichotomy may be due to differences in the molecular genetic environment of the tumor cells. BACKGROUND DATA: Diagnostic biopsies were obtained from a training cohort of esophageal cancer patients (13), and extracted RNA was hybridized to genome expression microarrays. The resulting gene expression data was verified by qRT-PCR. In a larger, independent validation cohort (27), we examined differential gene expression by qRT-PCR. The ability of differentially-regulated genes to predict response to therapy was assessed in a multivariate leave-one-out cross-validation model. RESULTS: Although 411 genes were differentially expressed between normal and tumor tissue, only 103 genes were altered between responder and non-responder tumor; and 67 genes differentially expressed >2-fold. These included genes previously reported in esophageal cancer and a number of novel genes. In the validation cohort, 8 of 12 selected genes were significantly different between the response groups. In the predictive model, 5 of 8 genes could predict response to therapy with 95% accuracy in a subset (74%) of patients. CONCLUSIONS: This study has identified a gene microarray pattern and a set of genes associated with response to neoadjuvant chemoradiation in esophageal cancer. The potential of these genes as biomarkers of response to treatment warrants further investigation. Copyright © 2009 by Lippincott Williams & Wilkins.

Facebook, telecollaboration, and international access to technology in the classroom

This study uses the well-known social networking site, Facebook (FB), for a study of differences in perceptions on the use of technologies in the classroom around the world. This study is part of a larger project exploring telecollaboration and the use of online discussions between graduate students in an online masters program based in Australia and students in the graduate education program at a regional university in Greece. Postings reveal more similarities between the situations and perceptions of the participants from the different countries than differences. Most participants indicated that while they and their students had access in general to computers and the internet, they did not necessarily have this access in the classroom. Even when technologies were available in schools, participants identified a critical need for professional development to increase teachers’ use of ICT. These findings are relevant to educators and policy development in terms of implementation of ICT or social networking in the language classroom.

Transparency tools in gene patenting for informing policy and practice

In the recent decision Association for Molecular Pathology v. Myriad Genetics1, the US Supreme Court held that naturally occurring sequences from human genomic DNA are not patentable subject matter. Only certain complementary DNAs (cDNA), modified sequences and methods to use sequences are potentially patentable. It is likely that this distinction will hold for all DNA sequences, whether animal, plant or microbial2. However, it is not clear whether this means that other naturally occurring informational molecules, such as polypeptides (proteins) or polysaccharides, will also be excluded from patents. The decision underscores a pressing need for precise analysis of patents that disclose and reference genetic sequences, especially in the claims. Similarly, data sets, standards compliance and analytical tools must be improved—in particular, data sets and analytical tools must be made openly accessible—in order to provide a basis for effective decision making and policy setting to support biological innovation. Here, we present a web-based platform that allows such data aggregation, analysis and visualization in an open, shareable facility. To demonstrate the potential for the extension of this platform to global patent jurisdictions, we discuss the results of a global survey of patent offices that shows that much progress is still needed in making these data freely available for aggregation in the first place.

The role of Y-box binding protein 1 in prostate cancer

This thesis examined the possible role of Y-box binding protein 1 (YBX1) in prostate cancer aggression and spread. Novel roles were uncovered for YBX1 in the regulation of several genes previously implicated in prostate cancer, as well as showing an effect for YBX1 in increasing tumour cell invasion and movement and reciprocal regulation of androgen-regulated gene networks. In addition, it was found that Y-box 1 regulated several other well-known cancer genes implicated in breast and other cancers. The work performed in this thesis has strengthened the foundations for pursuing YBX1 as a possible central target molecule in prostate cancer therapeutics.

Redistribution and technology adoption under uncertainty : a political economy perspective

We examine the role of politico-economic influences on macroeconomic performance within the framework of an endogenous growth model with costly technology adoption and uncertainty. The model is aimed at understanding the diversity in growth and inequality experiences across countries. Agents adopt either of two risky technologies, one of which is only available through financial intermediaries, who are able to alleviate some of this risk. The entry cost of financial intermediation depends on the proportion of government revenue that is allocated towards cost-reducing financial development expenditure, and agents vote on this proportion. The results show that agents at the top and bottom ends of the distribution prefer alternative means of re-distribution, thereby effectively blocking the allocation of resources towards cost-reducing financial development expenditure. Thus political factors have a role in delaying financial and capital deepening and economic development. Furthermore, the model provides a political-economy perspective on the Kuznets curve; uncertainty interacts with the political economy mechanism to produce transitional inequality patterns that, depending on initial conditions, can unearth the Kuznets-curve experience. Finally, the political outcomes are inefficient relative to policies aimed at maximizing the collective welfare of agents in the economy.

Luxury brand identity : the influence of mobile digital technology

This thesis examined the influence of mobile digital technology on the brand identity of luxury brands. Specifically it focused on the use of mobile applications by automobile, hotel and beauty brands and compared the perceptions of marketing managers with consumers on how mobile applications influenced luxury brand identity and image. Outcomes of this research included a model to depict the ongoing process between mobile-mediated luxury brand identity and image, and a typology of luxury brand mobile applications listing key features of mobile-mediated luxemosphere. Overall findings suggest that the influence of mobile applications on luxury brand identity has been negative, as their brand image appeared to be degraded, resulting in diminishing the brand identity.

Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis

In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission. © 2007 by The National Academy of Sciences of the USA.

Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of doublestranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5′ end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 59 thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.

RNA silencing in plants: Yesterday, today, and tomorrow

RNA silencing has become a major focus of molecular biology and biomedical research around the world. This is highlighted by a simple PubMed search for “RNA silencing,” which retrieves almost 9,000 articles. Interest in gene silencing-related mechanisms stemmed from the early 1990s, when this phenomenon was first noted as a surprise observation by plant scientists during the course of plant transformation experiments, in which the introduction of a transgene into the genome led to the silencing of both the transgene and homologous endogenes. From these initial studies, plant biologists have continued to generate a wealth of information into not only gene silencing mechanisms but also the complexity of these biological pathways as well as revealing their multilevel interactions with one another. The plant biology community has also made significant advancements in exploiting RNA silencing as a powerful tool for gene function studies and crop improvements. In this article, we (1) review the rich history of gene silencing research and the knowledge it has generated into our understanding of this fundamental mechanism of gene regulation in plants; (2) describe examples of the current applications of RNA silencing in crop plants; and (3) discuss improvements in RNA silencing technology and its potential application in plant science.