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Premier Lecture Series

Tissue microarray technology has emerged as a powerful tool for measuring tumor-specific protein expression and linking it to clinical outcome. Automated quantitative analysis of the tissue spots take the technology one step further, removing observer bias, providing standards for quality control and providing for high throughput analysis. We have developed a platform (PM1000) and software (AQUA) that allow rigorous in situ assessment of protein expression with the accuracy of an ELISA assay. We have used this platform on a series of large cohort tissue microarrays to discover a range of relationships between protein expression and outcome that are not discernable using conventional pathologist based analysis. We believe this sort of quantitative, objective validation will facilitate the translation of tissue biomarkers from the research lab to broad clinical usage.

Microarray (HGSM). The system uses 60mer oligonucleotides and chemiluminescence for detection increasing the sensitivity and specificity of expression. This was experimentally confirmed by a comparison of HGSM data and Real-Time RT-PCR. Pearsoncorrelation of 0.92 to 0.63 was found for main cancer genes, e.g. EGFR, Her2/neu, estrogen receptor α, progesterone receptor, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1. In order to study pathways by microarray analysis which represent distinct stages of differentiation specific signalling, esp. those regulating cancer cell invasiveness and motility, we established cellular models expressing EGFR, Her2/neu and genetic modifications by vectors expressing an truncated and an perturbed Her2/neu defect receptor as well as intracellular Her2/neu signalling inhibitors. All cell lines have been evaluated for phosphatidylinositol-and inositolphopshate signaling, PLC-γ1 activation, Ca2+-oscillation, actin reorganization and 3-dimensional migration. Applying a new biomathematical approach, we identified stable subsets of genes involved in the phosphatidylinositolphosphate and inositolphosphate metabolism, that explain the switch in cellular differentiation, consequently to a motogenic phenotype. For control the microarray data of the invasive breast cancer cellular models and genetically modified counterparts were re-analysed for the expression level and the significance of the difference in expression for the key molecule Her-2/neu simultaneously with all other genes. Two way hierarchical clustering of the expression data from these genes in breast cancer tissues from patients with complete clinical records clearly demonstrated distinct groups of cellular differentiation and clinical outcome. Therefore, our approach might be used as a classification tool to sort cancer into various clinically relevant subgroups that is not currently possible with other methods. The analysis of gene expression patterns may also be used to screen for individual genes that are differentially expressed between cancer tissues that respond and do not respond to a given therapy regimen.
In conclusion, the novel Human Genome Survey Microarray generates highly reliable and accurate data when performing whole genome expression profiling which might become a useful tool for clinical diagnostics.

-11.00
Dr Hilary Russell Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK

SEPTINS, THE CYTOSKELETON AND CANCER
Septins are an evolutionarily conserved group of GTPbinding and filament forming proteins that belong to the large superclass of P-loop GTPases. While originally identified in yeast as cell division cycle mutants with cytokinesis defects, they are now known to have diverse cellular roles which include polarity determination, cytoskeletal reorganization, membrane dynamics, vesicle trafficking, and exocytosis. Septin proteins form homoand hetero-oligomeric polymers which can assemble into higher order filaments. They are known to interact with components of the cytoskeleton, i.e. actin and tubulin. There are at least 12 human septin genes, and although information on expression patterns is limited, most undergo complex alternative splicing with some degree of tissue specificity. One model suggests that septins act as regulatable scaffolds where the stoichiometry of septin associations, modifications, GTP status, and the interactions with other proteins allow the regulation of key cellular processes. An increasing body of data implicates the septin family in the pathogenesis of diverse disease states including neoplasia, neurodegenerative conditions, and infections. Our research has focused on the SEPT9 gene which encodes up to 18 transcripts. We have shown that ovarian epithelial neoplasia is associated with increased levels of individual transcripts in particular SEPT9_v1 and_ v4*. We are therefore interested in determining the functional consequences of altered SEPT9 expression in epithelial cells and how this might contribute to aspects of the neoplastic phenotype. Evidence will be provided that SEPT9 can modulate remodelling of both the actin and microtubule components of the cytoskeleton. We find that SEPT9 overexpression causes enhanced motility, decreased polarity and resistance to microtubuleinteracting drugs -phenotypes associated with neoplasia.

Biography
Hilary Russell studied Genetics and Microbiology at Queen's University Belfast and completed her PhD in Biochemistry in 1984. After a period of postdoctoral research in the Department of Genetics, Trinity College Dublin, she returned to Queen's and established her own research group in 1988. She is currently a Senior Lecturer in Oncology in the Centre for Cancer Research and Cell Biology. The focus of her research is the molecular aetiology of epithelial ovarian cancer in particular, the role of the SEPT9 gene which the group identified by positional cloning. She also has a strong interest in research ethics and is Vice-Chair of one of the three Research Ethics Committees in Northern Ireland. Genetic alterations and expression changes are known to be associated with the neoplastic process. Recently developed tools enable the genome wide evaluation of genetic copy number and known gene and novel gene expression changes from the minute lung samples available as part of lung bonchoscopic examinations. Additionally advances in automated microscopy have enabled the automatic identification of cancer and precancerous cells in stained sections. Methods: 1)A CGH array designed to provide complete overlapping coverage of the human genome was used to examine genetic changes in neoplastic lung tissue.

References
2)SAGE was used to examine expression changes seen in a large set (40+) of lung tissue libraries. Selection of material for these technologies requires an estimate of the purity of the samples. 3)A lung pathology workstation for the analysis of preinvasive neoplastic bronchial samples has been adapted to estimate the percentage of neoplastic cells in H&E stained sections. 4)Cytology microarrays were used to evaluate the expression of some of the identified gene candidates from 1) and 2). Results: Arrays of ~30K amplified BAC DNA clones spanning the whole genome has been constructed. The impact of tissue heterogeneity and DNA quality/quantity in the generation of an accurate CGH profiles have been assessed. CGH profiling of a panel a collection of microdissected lung neoplastic specimens identified recurrent alterations. The repeatability and robustness of SAGE on limited lung samples has been demonstrated. More than 500 14mer tags of which 411 mapped to genes were found to be more than 50x over expressed between normal and cancer tissue. A small subset of the possible screening markers have been evaluated on a set of cytology microarrays constructed from 60 BAL samples from subjects with and without lung cancer. The performance of a few of these markers approach that required for a usable screening test.
O2 11.45 -12.00 Introduction. Long-term exhibition of the epithelium to factors such as tobacco and alcohol induces morphologic changes, ranging from pre-neoplastic hyperplasia and dysplasia to carcinoma in situ and invasive squamous cell carcinoma. This process is accompanied by the accumulation of genetic abnormalities. Gene copy number gains and losses were studied in 32 premalignant and 10 'normal' mucosa samples of the larynx using multiplex ligation probe amplification (MLPA). The aim was to find genetic changes involved in early stages of tumor development. Bayesian methods have been widely applied to data analysis problems in the life sciences. Their application to copy number change detection has however been limited to the post processing of primary data extracted from microarray images by more traditional block box image processing techniques. This combination of approaches is sensitive to variability; both from experimental and operator sources, in the primary data. In this presentation we describe a novel approach which combines the three major stages of arrayCGH analysis; extraction of primary data, normalisation and copy number change detection, in a single integrated solution. The Bayesian model based approach is introduced as a means of improving the measurement of the underlying biological quantities while at the same time estimating the likely variability associated with those measurements. The importance of a single statistical framework as a means of carrying forward knowledge of this variability through to the detection of copy number changes is illustrated by examples of how detected copy number changes may be qualified by confidence estimates; meaningful within the clinical context. This presentation will also summarise the experiment undertaken in the pre-conference workshop organised by Bauke Ylstra. This will illustrate how an integrated Bayesian approach removes operator intervention from the analysis of arrayCGH data thereby making this powerful technology accessible to individuals without a strong background in microarray image analysis and the associated mathematical techniques. Introduction. Chromosomal instability (CIN) is the most prevalent type of genomic instability in solid tumours. The resulting chromosomal aberrations affect genes in molecular pathways that are critical to tumour biology. One of these is gain of 20q, which frequently occurs in adenocarcinomas of different origin. 20q gain is associated with progression from colorectal adenoma to carcinoma, and is related with poor clinical outcome in gastric, breast, and colorectal cancer. We aimed to identify DNA copy number changes at gene resolution on chromosome 20q in a series adenocarcinomas of different origin in order to elucidate which genes and pathways are affected. Materials and methods. We analysed a total of 59 adenocarcinomas (35 colorectal, 11 gastric, 7 Barrett's oesophagus and 6 breast carcinomas) that showed 20q gains by CGH. The analysis was done by MLPA using a dedicated 20q probe mix.

PREMALIGNANT LARYNGEAL TUMORS AND APPARENTLY NORMAL MUCOSA FROM HEAVY SMOKERS SHOW RECURRENT GENE COPY NUMBER LOSSES AS REVEALED BY MLPA
Results. All tumours presented 20q gains as expected. Two amplification areas were detected, one at 20q11.2 and the other at 20q13.2. Genes involved in these amplified regions include BCL2L1 and ZNF217, respectively. ZNF217, a transcription factor, was amplified in all tumour types, but BCL2L1, which has role in apoptosis, was exclusively amplified in colorectal tumours.

Conclusion.
In conclusion, ZNF217 is amplified in adenocarcinomas from different origins and is likely to be a key player in carcinogenesis. Moreover, BCL2L1 seems to play a specific role in colorectal tumourigenesis. Introduction. Uncoupling of cell cycle and mitotic checkpoints and inhibition of apoptosis may lead to chromosomal instability (CIN) and aneuploidy. Along with these hypotheses, we investigated the role of KRAS2 and APC mutations in CIN using human sporadic colorectal adenomas (hscAD).
Methods. Multiple samples were obtained from 185 hscAD to provide nuclei suspensions for flow-cytometric analysis of the DNA Index (DI) and isolated DNA for DNA sequencing of KRAS2. In a subgroup of 85 hscAD, morphologic analysis was done to count atypical mitoses, cells with apoptotic bodies, and to separate architectural from cytokarylogic dysplasia. APC mutation spectrum was additionally evaluated by DNA sequencing in 65 hscAD.
Results. KRAS2 and APC mutations and DNA aneuploidy (DI&#8800;1) were detected respectively in 42/185 (23%), 20/65 (31%) and 59/185 (32%) cases. Aneuploidy was characterised by near-diploidy (DI<1.4) in the majority of cases. Incidence of aneuploidy among KRAS2 wild-type, G--A transitions, and G--C/T transvertions were respectively 29%, 22% and 63%. KRAS2 G--C/T transvertion mutations were strongly associated with aneuploidy as proven by contingency table-analysis (pFisher<0.005) and by multivariate logistic regression to account for the effects of size, dysplasia, site, type, age and sex (Odd-Ratio=6 ). G--C/T transvertions were also associated with a lower level of apoptosis (pChi-Square=0.01), while KRAS2 mutational status was associated with cytokarylogic dysplasia (pFisher=0.02). We have additionally observed association of the APC specific mutations within and downstream the mutation cluster region (&#8805;MCR) with aneuploidy (pChi-Square=0.006). The corresponding truncated proteins with respect to the proteins for mutation upstream MCR (approximately at codons lower than 1200) are characterised by the partial conservation of Beta-catenin binding and degradation sites. Multivariate analysis for APC to account for covariates awaits additional cases.
Conclusions. These data suggest that specific KRAS2 and APC mutations in subgroups of hscAD play a role in chromosomal instability and aneuploidy. Cell clones overexpressing full-length DDR1 responded with sustained receptor activation upon collagen stimulation. We found that the expression of all three different isoforms increased the adhesion of MDA-MB-231 cells to collagen approximately 1.4 fold compared to mock-transfected cells, while cell proliferation remained unchanged. To examine its in vivo function in immunocompromised mice, DDR1-overexpressing cells were cotransfected with EGFP and either intracardially injected or orthotopically implanted into to the mammary fat pad. For a period of one month, tumor cell growth and dissemination as well as the formation of bone metastases was monitored twice per week in anaesthetized animals using the Kodak Image2000 station. We observed a higher incident of bone tumors in mice injected with cells overexpressing DDR1b than control-transfected cells.
Volumetric computed tomography (VCT) as well as Faxitron analysis were used as alternative methods to quantify the extent of tumor load and metastasis. Conclusion. Our data provide compelling new evidence that DDR1 is involved in basement membrane attachment and invasion of human breast tumor cells.

DEATH AND ANTI-DEATH
CD95, a member of the tumor necrosis factor (TNF) receptor superfamily induces apoptosis upon receptor oligomerization. The receptor and its ligand are important for apoptosis of peripheral T cells, for downregulation of an immune response and most likely, at least in part, also for peripheral T cell tolerance. In AIDS, apoptosis mediated by this system might contribute to the depletion of T helper lymphocytes. Likewise, in diseases in which liver cells are destroyed the CD95 system might play a major measurement of protein analytes on the surface of a biochip, which results in the specific and simultaneous profiling of protein markers. Rather than having to divide and separately test a patient sample to obtain each test result, biochip array technology offers a means for simultaneous testing of a sample and thus provides a more complete diagnostic profile for each patient.