research report

University of Leeds

2001/02

Research School of Medicine
Director: Professor M.A. Smith

Tumour vascularity, cellular proliferation and the role of angiopoietins in colorectal cancer
Mr D Burke, Prof. P. Quirke and Dr P.F. Jones

Colorectal cancer is the second most common cause of cancer in the UK, with approximately 28000 new cases per annum. Although the genetic development of this malignancy has been fairly well defined, the biological processes involved are poorly understood. It is now established that tumours cannot grow beyond 1-2mm in size without the development of an adequate blood supply. This process is termed angiogenesis. However, the effect of angiogenesis upon basic malignant cell biology such as tumour cell division, apoptosis, metastasis and nutrition is not known and warrants further study.

Angiogenesis is stimulated by a variety of tissue proteins, acting via kinase domain-containing receptors. Recently there has been described the Angiopoietin family that acts via a unique, Tie-2, receptor. There is evidence within other tumour types that the angiopoietins may be an early marker of tumour vessel development and that subsequent vessel development may depend upon the balance of these proteins.

In collaboration with Professor P. Quirke (Dept of Pathology) and Dr P. Jones (Molecular Medicine Unit) we are developing techniques to examine colorectal cancer specimens for the presence of Angiopoietins 1 and 2, together with the Tie-2 receptor. These specimens will similarly be assayed for the cellular proliferation and apoptosis rates. These investigations will allow us to determine whether Angiopoietins are potentially important in the development of colorectal cancer and whether the tumour vascularity might influence tumour growth.

Activation of the GM-CSF locus in acute myeloid leukaemia
Dr P.N. Cockerill

GM-CSF is an inducible growth factor that normally supports the growth of myeloid lineage white blood cells. The GM-CSF gene is also known to be inappropriately activated in a proportion of acute myeloid leukaemias (AMLs) that are dependent upon the autocrine synthesis of GM-CSF for their autonomous growth. We are investigating mechanisms that contribute to the normal inducible regulation of the GM-CSF gene in white blood cells, and to the abnormal constitutive activation of the GM-CSF gene in AML.

We have identified an inducible transcriptional enhancer 3 kb upstream of the GM-CSF gene. This enhancer is required for GM-CSF gene activation in myeloid cells. When normal myeloid cells are activated, the chromatin structure of the enhancer is remodelled and a DNaseI hypersensitive site (DHS) is formed. In several AMLs that we have studied, the enhancer exists as an aberrantly activated constitutive DHS. To study mechanisms involved in the formation of this DHS we have used in vivo footprinting to identify bound transcription factors. Starting with normal cells we have demonstrated that 2 sites within the DHS are rapidly occupied by NFAT following activation. NFAT appears to function as a chromatin remodelling factor in this locus, and other factors required for enhancer function only bind after NFAT binding and chromatin remodelling has occurred. One of the factors that binds the enhancer subsequent to NFAT binding is AML1, the product of a proto-oncogene that contributes to AML.

Tumour initiation and progression in the ApcMin mouse model of familial adenomatous polyposis
Dr P.L. Coletta, Dr C. Bonifer and Dr M.A. Hull

The Adenomatous polyposis col (Apc) tumour suppressor gene is mutated in hereditary and sporadic forms of colorectal cancer and in the ApcMin mouse model of intestinal tumorigenesis. Loss of normal Apc function in intestinal epithelial cells is followed by the accumulation of mutations in a number of tumour suppressor and oncogenes, which drives progression along the adenoma-carcinoma pathway. We have previously shown that the inducible form of cyclooxygenase, Cox-2, is upregulated in macrophages early in this pathway in the ApcMin mouse and in human sporadic colorectal adenomas. The reason for the upregulation of Cox-2 is however not known. We are investigating the mechanism by which Cox-2 is upregulated specifically in macrophages and the functional consequences of changes in macrophage gene expression.

We are using in vitro and in vivo methods to compare intestinal permeability in ApcMin and wild-type littermates. This approach will allow us to determine whether or not mutations in Apc affect intestinal permeability and barrier function leading to Cox-2 expression in macrophages. We are also using a transgenic model in which macrophages are labelled with Enhanced Green Fluorescent Protein (EGFP) to investigate macrophage gene expression and function in intestinal tumorigenesis. An understanding of the molecular mechanisms that promote colorectal tumorigenesis will facilitate development of novel therapies and chemopreventative strategies for colorectal cancer.

The role of Helicobacter pylori cag locus and host inducible nitric oxide synthase on gastric epithelial cell function and mucosal damage.
Dr J.E. Crabtree, Dr P.A. Robinson, Prof. M.F. Dixon, Miss M. Court

Infection with Helicobacter pylori strains containing the cag pathogenicity island (cag PAI) is strongly associated with development of gastric atrophy and intestinal-type of gastric cancer. The expression of apoptosis related genes in gastric epithelial cells differs following stimulation with cag+ and cag- strains. The aim of the study was to identify the effects of cag virulence genes on epithelial biology in vivo in H. pylori models. Long term infection studies with cag PAI positive and cag PAI negative H. pylori strains have characterised the effects of strain variation on gastric pathology, epithelial cell proliferation and apoptosis. To investigate the role of inducible nitric oxide synthase (iNOS) in apoptosis and Helicobacter induced mucosal damage, homozygous mutant mice lacking iNOS have been infected with gastric Helicocbacter for up to 12 months. The effects of infection on gastric pathology, epithelial cell proliferation and apoptosis was assessed. Gastric epithelial hyperplasia, oxyntic cell loss, epithelial proliferation and apoptosis were markedly increased in mice lacking iNOS compared to wildtype mice. Cyclooxygenase 2 was upregulated in the gastric mucosa of infected wildtype mice but not in mice lacking iNOS. This suggests that the upregulation of iNOS which occurs in Helicobacter infection has a protective role in Helicobacter induced gastric pathology. Identification of changes in epithelial cell function induced by the cag PAI and iNOS should improve our understanding of the role of H. pylori in mucosal damage and gastric carcinogenesis.

Characterisation of host genes associated with the development of Helicobactor pylori induced gastric cancer
Dr J.E. Crabtree, Dr M. Aboshkiwa, Prof. M.F. Dixon, Dr P.A Robinson

Like most cancers, gastric cancer occurs through a multistage process. H.pylori has been recently classified as a category I human carcinogen playing a causative role in the development of gastric cancer. Long-term infection of an in vivo model with H.pylori results in the development of gastric cancer. This model has been established to permit identification of H.pylori induced host genes during gastric cancer progression. Long term infection studies are in progress. Our previous study using cDNA array technology identified hundreds of known and novel host genes which were differentially expressed in gastric epithelial cells following stimulation with H. pylori. H. pylori upregulates both epidermal growth factor (EGF) ligands, such as amphiregulin and heparin binding-EGF, and also several genes encoding for ADAM metalloprotease-disintegrin proteins in gastric epithelial cells. We have demonstrated that EGF receptor transactivation induced by H. pylori is dependent on extracellular transmembrane metalloprotease cleavge of proHB-EGF and signalling via mature HB-EGF. This signalling pathway is likely to lead to autocrine/paracrine signalling cascades promoting enhanced gastric epithelial cell proliferation and decreased apoptosis and ultimately neoplasia. We have identified that the expression of several members of the ADAMs family are increased in patients with H. pylori infection and also in gastric cancer. To investigate the importance of H. pylori induced EGF receptor signalling in the model system, ADAMs genes and HB-EGF have been characterised in the model. The gastric expression of these genes and relationship to bacterial induced changes in gastric epithelial proliferation, apoptosis and development of neoplasia is being examined. The model system will allow future assessment of interventional strategies.

The role of free radical induced DNA damage during the evolution of oesophageal adenocarcinoma
Dr L.J. Hardie, Mr G.W.B. Clark, Prof. C.P. Wild

The incidence of oesophageal adenocarcinoma (OA) is increasing by 10% per annum, surpassing the rate of increase for any other cancer type in western societies. OA is believed to develop from the pre-malignant condition, Barrett's oesophagus, where the normal squamous epithelium of the oesophagus is replaced with a specialised intestinal type, usually in response to severe heartburn or gastro-oesophageal reflux.

The early molecular mechanisms driving the development of Barrett's oesophagus and OA are currently unclear. We are testing the hypothesis that gastro-oesophageal reflux stimulates oxidative stress and DNA damage in Barrett's oesophagus rendering this tissue prone to malignant transformation.

Over the last year we have collected oesophageal biopsies from Barrett's oesophagus and dyspeptic control patients and analysed these for levels of DNA damage. Data collected to date reveal significantly increased levels of DNA damage in Barrett's mucosa compared with the normal squamous mucosa of these patients, and is consistent with a role for oxidative DNA damage during this disease process. Over the next year, we will recruit further control, Barrett's and oesophageal adenocarcinoma patients, with a view to understanding the factors which modulate DNA damage levels in oesophageal tissue. In addition to providing an insight into the natural history of this disease process, levels of DNA damage may help us identify those patients at increased risk of disease progression and serve as a biomarker to assess the efficacy of therapeutic regimes.

Down-regulation of b-catenin underlies chemoprevention of colorectal cancer by non-steroidal anti-inflammatory drugs
Dr G. Hawcroft, Prof. A.F. Markham and Dr M.A. Hull

Non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin have a significant but poorly understood protective effect against colorectal cancer (CRC). We have recently demonstrated that, in human CRC cells in vitro, indomethacin decreased ?-catenin and TCF target genes (including cyclin D1) expression. We are now investigating whether other NSAIDs decrease expression of ?-catenin and TCF target genes in vitro. We are also investigating whether regular NSAID use reduces ?-catenin expression in colorectal adenomas taken from patients. Confirmation of the hypothesis that inhibition of ?-catenin signalling is an important element of the anti-neoplastic activity of NSAIDs will enable us to select NSAIDs, with the highest efficacy in this regard, for early clinical evaluation as chemopreventative agents for CRC.

GPI deficient lymphocyte function and opportunistic infection in patients with chronic lymphocytic leukaemia treated with CAMPATH-1H
Dr P. Hillmen and Dr B. Kennedy

CAMPATH-1H is a monoclonal antibody specific for the glycosylphosphatidylinositol (GPI) anchored CD52 molecule. We have treated almost 50 patients in the Yorkshire region who have refractory B-CLL. We have shown GPI-anchor deficient cells form the majority of reconstituting CD3+ T-lymphocytes after CAMPATH-1H therapy in 75% of cases. Using 4 colour flow cytometry we have monitored T-cell recovery after CAMPATH-1H. Median CD3+ count during CAMPATH-1H therapy is < 0.01x109/l. At the end of therapy a median of 97% CD3+ cells are GPI deficient. We have reported an 8.9% risk of reactivation of CMV infection, which is associated with significant mortality. Using a CD2/CD2R invitro activation system we found that GPI-deficient T-cells demonstrate normal upregulation of CD69. We found no change in expression of IL2 receptor, CD71 or HLA DR after activation. An increased proportion of GPI-deficient CD3+ cells express the CD45ROHigh memory phenotype. We have isolated T cells before and after Campath therapy by magnetic columns and performed T cell subset analysis, for naïve cells, by flow cytometry. We have in collaboration with Dr J. Isaacs, performed real time semi-quantitative PCR to identify early thymic emigrants by measuring T cell receptor excision circle, (TREC) on these selected naïve cells. Analysis of data is ongoing. We have analysed T-cell receptor V? expression by flow cytometry. Our data show that GPI-deficient T-cells are polyclonal and the range of TCRV? appears restricted. The clinical use of CAMPATH-1H is accelerating rapidly. Our studies have prompted wide interest.

The role of cyclooxygenase-2 (COX-2) in intestinal tumorigenesis
Dr M.A. Hull, Dr C. Bonifer and Dr P.L. Coletta

We have investigated expression of the inducible isoform of cyclooxygenase-2 (COX-2) at different stages of human colorectal carcinogenesis from the benign colorectal adenoma to metastatic colorectal adenocarcinoma of the liver.

Direct genetic evidence suggests that COX-2 plays an important role in the early stages of intestinal tumorigenesis. We have previously demonstrated that COX-2 is expressed predominantly in interstitial (or stromal) macrophages in intestinal adenomas. We have now developed a model of macrophage-specific COX-2 over-expression in order to further investigate the putative paracrine mechanism by which interstitial macrophage COX-2 promotes intestinal tumorigenesis.

Investigation of the role of COX-2 in the early stages of intestinal tumorigenesis may lead to the development of safe and effective chemopreventative therapy for colorectal cancer.

Identification of New Angiopoietins
Dr P.F. Jones and Prof. A.F. Markham

The identification of novel factors involved in tumour growth and development is critical for our understanding and treatment of cancer. The angiopoietins, ligands for the endothelial cell-specific receptor tyrosine kinase Tie2, represent a family of angiogenic factors critically involved in the growth and maintenance of vasculature. Data obtained from transgenic mice deficient for angiopoietin-1 suggest the existence of still further members of this family.

Conventional low-stringency cloning methods have revealed several molecules showing homology to the angiopoietins, but which do not bind Tie2, implying they are not true ligands for this receptor. Similarly, we have developed PCR-based screens for further distant family members, which have not revealed any novel factors that bind to Tie2. Further orthologs of the angiopoietin family have been identified. Furthermore, reagents developed during these low homology screens have enabled more detailed characterisation of the genes encoding the three previously identified human angiopoietins. Interestingly, although no polymorphism has yet been identified in Ang1, Ang2 appears to be highly polymorphic, with three independent polymporphisms identified thus far within the coding region. Given the role of Ang2 in tumour angiogenesis, it will be interesting to determine any correlation between tumour type or metastatic potential and these polymorphisms.

Investigation and characterisation of genetic abnormalities associated with illegitimate isotope switching rearrangements at 14q32 in multiple myeloma patient material
Prof. G.J. Morgan

Studying the genetics of multiple myeloma has implications for improving our biological understanding of the disease process and how we may improve treatment.

Clinically we have continued to investigate PCR and flow cytometric monitoring of disease response in myeloma. In addition we are exploring how genetic variants in gene families, which modify the effects of chemotherapeutic agents, can affect response to treatment. In particular the role played by GSTP1 is being investigated.

We have used PCR based approaches to look at the mechanisms underlying the common chromosomal translocations in myeloma: Current results suggest that aberrant class switch recombination targeted to the 5´ region of the switch region sequences is a critical process. Detecting the consequences of these translocations can be used diagnostically and for monitoring treatment.

The role of fibroblast growth factor receptor 3 (FGFR3) in multiple myeloma using an in-vitro model system
Prof. G.J. Morgan

We are initiating a project looking at the effects of over expressing of the tyrosine kinase receptor FGFR3 at different stages of haemopoietic differentiation. This gene is commonly deregulated in myeloma and these experiments will allow us to understand its effects in cell growth and differentiation as well as the downstream genes which mediate these effects.

Interaction between the APC tumour suppressor protein and the mitotic spindle component EB1 and the significance of its disruption in colorectal carcinoma.
Dr E.E. Morrison and Dr J.M. Askham

Mutation of the APC gene is a frequent event in colorectal carcinoma. These mutations commonly result in the production of a truncated protein which has lost the ability to interact with a number of partners. One of these is a microtubule tip-associated protein called EB1. We have previously shown that EB1 also interacted with the p150glued subunit of the dynein/dynactin microtubule motor complex, and that the region in EB1 responsible for this binding overlapped with that required for the APC interaction. We have now confirmed that EB1 cannot simultaneously bind to p150glued and APC. We have localised the EB1 binding site in p150glued to the extreme N-terminus of the protein, a region which contains a number of phosphorylation sites, and are now investigating whether these sites regulate the interaction between EB1 and p150glued. In addition, we have exploited our previous observation that EB1 specifically localises to growing microtubule tips to address a number of basic issues in cell biology. Specifically, we have used EB1 immunofluorescence and live imaging of EB1-GFP to quantitatively investigate sites of microtubule growth in neuronal axons. Furthermore, we have used EB1 immunofluorescence to quantitatively examine growing astral microtubules during different stages of mitosis. This revealed a sudden increase in astral microtubule length and number at anaphase onset, and we went on to show that this was essential for the correct positioning of the mitotic spindle in dividing cells. Ongoing studies are directed towards examining whether EB1 itself might play a role in this process.

Investigation of the role of TOGp in colorectal cancer
Dr E.E. Morrison, Dr J.M. Askham and Mr M. Adams

TOGp is a large microtubule-associated protein which is overexpressed in colorectal and hepatic carcinoma. Based on studies of its homologs in other organisms, it is thought to play a major role in promoting microtubule growth, particularly during mitosis. Using confocal microscopy and an antibody kindly provided by Dr Duane Compton, Dartmouth Medical School, USA, we have examined the distribution of TOGp within cells. We found that TOGp colocalised with EB1 at microtubule tips in interphase cells, suggesting that it specifically localises to the tips of growing microtubules, consistent with its potential role in contributing to microtubule extension. In mitotic cells strong staining on kinetochore microtubules was seen from metaphase onwards, and on astral microtubules from anaphase onwards. TOGp was also observed at the centrosome in interphase cells and the spindle poles during mitosis. The TOGp cDNA has been cloned, and a number of expression constructs derived from this have been made as fusions to GST for bacterial expression (with a view to raising our own antibodies to the protein) and eGFP for expression in mammalian cell lines. To date, these have demonstrated that the centrosomal localisation of TOGp is mediated by the C-terminal third of the protein, and possible mechanisms by which this targeting is mediated are currently under investigation. Construction of a full-length TOGp-eGFP construct which will allow the behaviour of this protein to be examined in live cells is also underway, as are studies aiming to examine whether TOGp interacts with EB1.

Application of advanced image analysis technology to cancer cell biology
Dr E.E. Morrison

This equipment grant funding was used to purchase an SGI computer workstation running a suite of image analysis software from Bitplane AG, intended to complement and extend the capabilities of existing imaging systems on the St James's University Hospital and main University campuses. The system was installed in the summer of 2001 and it is currently directly linked to the confocal imaging system within the Centre for Molecular Medicine. It has been used extensively in much of the work recently performed on the confocal microscope, including the publications from our laboratory detailed in this report. As knowledge of and familiarity with the system increases, it is certain to play a larger role in the work of many of the investigators using imaging technology at the University of Leeds. Recognition of the utility of the equipment has been recognised by other users of the confocal imaging facilty, leading to the purchase of further Bitplane software by other researchers on the St James's campus.

Selection of patients for adjuvant therapy in colorectal cancer - getting it right with molecular profiling
Prof. P. Quirke, Dr M. Seymour

The study is making good progress. We have recently analysed Quasar 1 data for prognosis but not yet by treatment allocation which is not available until the trial closes. We have analysed 9 markers on 180 patients (p53 x3, 18q x4, 12 and 14) for LOH. p53 and D17ST86 (p<0.05) was the only variable with statistical significance for prognosis.

We have analysed over 600 patients for expression of thymidylate synthase, dUTPase, p53, hMLH1, hMSH and MGMT. There are significant associations with dUTPase (p0.005) and less significant associations with thymidylate synthase and hMSH-2. We await the response to therapy data becoming available.

The most powerful prognostic markers remain histopathological. Tumour stage, peritoneal involvement and extramural vascular invasion are all highly significant for prognosis in Quasar 1. We are currently making tissue microarrays and are beginning to quantitate the levels of expression of proteins.

This grant has contributed to the recent award of National Translational Cancer Research Centre status for Leeds and Bradford and towards the development of National Tissue Banking policies. Substantial publications are not expected until treatment data becomes available.

A collaborative Yorkshire approach to the localisation of genes involved in gastric cancer by sequential comparative genomic hybridisation and microsatellite screening using familial and young onset gastric cancer
Mr I. Martin, Dr D. Hammond, Dr J. Royds and Prof. P. Quirke

Our recent work has shown that patients with gastric cancer under the age of 40 demonstrate less numerical aberrations and apparently specific lesions of high level amplification of 8p, gain on 18p, loss on 8q and 11q providing sites for further exploration either by high resolution CGH or real time quantitative PCR. The low level of loss is similar to early onset colorectal cancers. Tissue microarrays of gastric cancer have been constructed on 200 cases and the possibility of quantitative FISH on TMAs at these loci is also under investigation to confirm the specificity of these lesions in young onset cases and the low frequency of these lesions in a series of sporadic cancer. We have also investigated 92 sporadic cases for 12 markers of allelic imbalance on chromosome 1p which was frequently lost in the sporadic cancers. The use of real time quantitative PCR for rapid assessment of loss and gain of sites is being evaluated but looks very promising as a rapid method of narrowing the areas of deletion and amplification. This would allow more rapid progress than microsatellite mapping.

The significance of estrogen receptor ? in breast cancer
Dr V. Speirs and Dr G. Skliris

Two human estrogen receptor (ER) isoforms exist, the "classic" ER? and the more recently identified ER?. ER? is an important prognostic factor in breast cancer, and although ER? is expressed in the breast, its biological role remains undefined. To gain insights into the possible role of ER? during breast carcinogenesis immunohistochemical analysis of ER? was performed on over 500 breast specimens encompassing the full spectrum of breast pathologies i.e. normal gland, ductal carcinoma in situ, invasive cancers, lymph node metastases and recurrences. A gradual reduction, but not a complete loss of expression of ER? was observed during the transition from normal breast and pre-invasive lesions (near constitutive expression) to invasive cancers where the receptor was lost in 20% of cases. This was more pronounced in invasive ductal compared to invasive lobular carcinomas where a significantly higher proportion were ER? positive. Our results suggest that loss of expression of ER? may be one of the hallmarks associated with breast cancer progression. We are now going on to determine if this is a reversible process perhaps involving hypermethylation of the ER? promoter.

Kaposi's sarcoma: investigating the Human Herpesvirus-8 latent-lytic switch
Dr A. Whitehouse and Dr D.J. Goodwin

Kaposi's sarcoma-associated herpesvirus or human herpesvirus 8 (HHV-8) is a lymphotrophic ?-2 herpesvirus associated with Kaposi's sarcoma (KS), a major neoplasm particularly in AIDs patients. KS is an endothelial neoplasm. However, the primary reservoir of latent virus is established in B lymphocytes. Reactivation to lytic HHV-8 infection from the latently infected lymphoid reservoir is essential for the spread of infectious virus to the endothelium. Therefore, reactivation from the latent state into the lytic cycle, allowing active HHV-8 gene expression and replication is a necessary antecedent step in KS development. However, little is known about the nature of the latent-lytic switch. We have demonstrated that the ORF 50 gene products in ?-2 herpesviruses are essential transcriptional regulators, activating the lytic replication cycle. Moreover, forced expression of this protein under the control of an heterologous promoter, has the ability to trigger reactivation of the lytic replication cycle from an established latent infection. This suggests that regulation of the ORF 50 promoter itself is a key component of the switch from latency to a lytic replication cycle and is an important determinant of the natural history of the viral infection. We are now performing detailed analysis of the ORF 50 promoter using DNase I footprinting. At present further work is being performed to determine whether a number of tentatively identified cellular factors play central roles in regulating the latent-lytic switch in gamma-2 herpesviruses.

Development of a replication-disabled Herpesvirus saimiri gene transfer vector for the delivery of therapeutic genes to lung carcinoma cells
Dr A. Whitehouse, Dr K.T. Hall and Prof. A.F. Markham

This study aims to evaluate the potential of Herpesvirus saimiri (HVS) as a novel gene therapy vector. This virus has the ability to accommodate large quantities of heterologous DNA, infect non-dividing cells and maintain its genome episomally in a non-replicative form. We have succeeded in producing a recombinant HVS expressing the Enhanced Green Fluorescent Protein (EGFP) and demonstrated that this virus can infect a range of human cancer cell lines, including lung carcinoma cells, and direct sustained production of an exogenous protein. We have optimised infections and determined the stability of the episome and expression characteristics of the marker gene. In addition, genes essential for the virus to proceed through the lytic replication cycle have been identified, namely the the two major transcription control genes encoded by the virus, ORF 50 and ORF 57, and replication-disabled viruses are in the process of being produced. Furthermore, in vivo analysis of HVS-based vectors have been performed demonstrating that HVS can persist as a circular non-integrated episome, which allows the expression of a transgene in an in vivo environment. These results suggest that HVS-based vectors have promising potential for gene therapy applications.

Development of an adenovirus vector which utilises the Herpesvirus saimiri episomal maintenance system to allow efficient long term expression of therapeutic tumour suppressor genes in carcinoma cells
Dr D.A. Matthews, Dr K.T. Hall, Dr A. Whitehouse

This study aims to develop an improved adenovirus cancer gene therapy vector. This will initially involve determination of the minimal components of the Herpesvirus Saimiri (HVS) episomal maintenance system. We have demonstrated that HVS will maintain episomes in a wide range of human, primate and murine cells. Thus the episomal maintenance elements will have broad utility for the delivery of therapeutic genes to a wide range of malignant cell types. We aim to combine this attractive feature with an already established gene therapy vector, adenovirus. Currently, this vector is not capable of maintaining a delivered transgene over successive rounds of cell division, a critical requirement when attempting to moderate tumour cell growth with tumour suppressor genes. We have now identified the minimal elements required for HVS episomal maintenance. These comprise a trans-acting factor encoded by ORF 73, which co-localises with host mitotic chromosomes and a cis-acting element contained with the terminal repeats of the HVS genome. Future work will involve combining the episomal maintenance elements into a recombinant adenovirus and characterise its utility to deliver and maintain a range of therapeutic transgenes in an episomal state over several rounds of cell division in carcinoma cells.

Identification of Barrett's oesophagus patients at high risk of progression to oesophageal adenocarcinoma using molecular markers
Prof. C.P. Wild, Prof. D. Forman, Dr M. Scott

Adenocarcinoma of the oesophagus (ADC) develops from the premalignant condition know as Barrett's oesophagus (BO). This change is accompanied by a series of molecular alterations in genes which regulate cell division. These alterations may be used to identify BO patients at particularly high risk of progression to ADC and refine endoscopic surveillance programmes. Using such an approach, we have already shown that cyclin D1 overexpression is associated with a seven fold increased risk of progression to ADC.

Over the last six months, we have identified all ADC cases which have arisen in Northern Ireland between 1993-1999. These patients have been closely matched to a series of BO patients who have not progressed to cancer over an equivalent surveillance period. All biopsies collected from both patient groups are currently being accessed from hospital tissue archives and subjected to a battery of tests for molecular markers, using protocols which have been carefully validated in the laboratory.

It is anticipated that information obtained from this study will confirm whether molecular markers can be used to refine entry criteria into endoscopic surveillance programmes and potentially offer earlier detection and more effective treatment of ADC.

Response to Neoadjuvant Therapy in Oesophageal Cancer: The Role of Survivin, A Novel Inhibitor of Apoptosis
D.M. Beardsmore, C. Verbeke, C. Davies, P.J. Guillou, G.W.B. Clark

Altered expression of the genes that control apoptosis and proliferation may influence how cancer cells respond to radiotherapy and chemotherapeutic agents. The aim of this study was to determine the role of the novel anti-apoptotic and cell-cycle gene, survivin, in apoptotsis and proliferation in esophageal cancer and to evaluate whether survivin, p53 and Bcl-2 status were able to predict a patient's response to neoadjuvant therapy.

104 esophageal tumours were studied. The cancer specimens were immunostained for survivin, p53 and bcl-2 proteins. Survivin expresion was graded (0-12) according to the percentage and intensity of stained cancer cells. P53 and bcl-2 expression was classified as positive or negative. Proliferative and apoptotic cells were measured using KI-67 and the TUNEL method respectively. The proliferative and apoptotic indices were expressed as a percentage after counting the number of immunostained cells per 1000 cancer cells. 48 patients whose pre-treatment biopsies were analysed underwent neoadjuvant chemoradiotherapy (40) or chemotherapy (8) followed by surgery. Outcome was graded as a complete response (CR), a partial response (PR) or no response (NR) according to histological examination and CT imaging.

Expression of survivin was found to correlate significantly with the proliferative index. Patients who received neoadjuvant treatment were more likely to receive a CR if their tumours had high proliferative activity or if they were p53 negative. Conclusions: Survivin expression appears to foster proliferative activity in esophageal cancer. In addition, this study shows that tumours with a high proliferative index and a functioning p53 gene are more responsive to neoadjuvant chemoradiotherapy

Interactions between mesothelial cells and tumour cells in the generation of peritoneal metastases
Prof. P.J. Guillou, Mr D. Jayne, Prof. A.F. Markham

Peritoneal metastases are common in patients with gastrointestinal cancer. The peritoneum is one of the commonest sites of tumour recurrence following curative resection. The objectives of this project were to identify the interactions which occur between mesothelial cells and tumour cells in the generation of peritoneal metastases. Preliminary work has demonstrated that under the conditions of high cytokine concentration, which is always found within the peritoneal cavity following surgical operation, the mesothelium is activated to produce certain heparin-binding growth factors to which gastrointestinal tumour cells frequently possess receptors. These growth factor receptor interactions are known to stimulate proliferation and invasion. Further studies have been conducted to categorise the repertoire of growth factors generated by mesothelial cells in response to cytokine activation. This repertoire includes HB-EGF and 2-VEGF, splice variants VEGF-121 and VEGF-165, but not basic FGF. Current studies are determining whether or not cytokines such as scatter factor (HGF) are also produced by the activated mesothelium, this work may lead to therapeutic manipulations which may prevent the development of peritoneal metastases following surgical resection of gastrointestinal cancers.

The role of apoptosis in peritoneal invasion
Prof. P.J. Guillou and Mr. D. Jayne

During the course of a series of projects aimed at identifying the interactions which occur between tumour cells and the mesothelium in the generation of peritoneal metastases, it was observed that co-culture of tumour cells with mesothelial cells resulted in the development of apoptotic bodies suggesting that in some way apoptosis was implicated in peritoneal metastasis development. Using the TUNEL assay, apoptosis in co-cultures was quantified and studied using confocal microscopy. The results indicated that whilst there was a very low level of apoptosis amongst tumour cells alone and amongst mesothelial cells alone, the co-culture resulted in massive induction of apoptosis amongst the mesothelial cells. This was related to the expression of death ligands and their receptors on corresponding tumour and mesothelial cells. Co-cultures which including neutralising antibodies to death ligands such as APO-1 (FasL) reduced the level of mesothelial apoptosis induced by tumour cells but did not eliminate it completely. Further studies are in progress to identify other death ligands which may be implicated in this process.

In order to determine whether this process truly influences peritoneal metastasis formation a three dimensional model of the human mesothelium has been developed. This consists of a collagen lattice upon which is grown a monolayer of human mesothelial cells. This structure has all the characteristics of the human peritoneum, including expression of ß1 Integrin subunits to which tumour cells adhere. This three dimensional model of the peritoneum has been seeded with various tumour cell lines which adhere to and invade through the mesothelial layer into the collagen lattice and produces 'metastases', which are histologically identical to those obtained from patients. This in vitro model is now being utilised experimentally to examine the processes whereby such metastases are generated, and to determine the role of different death ligands in the induction of mesothelial apoptosis which results in the formation of a metastasis.

Faculty of Biological Sciences
(School of Biochemistry and Molecular Biology)
Chairman of Faculty: Professor K.T. Holland
Head of the School: Professor A.J. Turner

Investigation into the role of Bcr-Abl-induced abnormalities in glucose transport regulation in the pathogenesis of chronic myeloid leukaemia
Dr U. Bugajska, Dr. K. Barnes, Ms E. McIntosh, Ms D. Itchayanan, Dr J. Bentley and Prof. S.A. Baldwin

Recent studies from several laboratories, including our own, have shown that a primary function of haemopoietic growth factors is to maintain sufficient rates of glucose uptake and metabolism to suppress mitochondrion-initiated apoptosis. Cell survival is thus ensured under appropriate circumstances. In contrast, expression of the chimaeric tyrosine kinase Bcr-Abl is associated with inappropriate survival of haemopoietic stem cells and is a key step in the development of chronic myeloid leukaemia (CML). Using the multipotent haemopoietic cell line FDCP-mix tsBcr-Abl as a model for chronic phase CML, we have shown that inappropriate suppression of apoptosis by the Bcr-Abl oncoprotein involves the abnormal maintenance of high rates of glucose uptake in the absence of growth factor. The stimulatory effect of the oncogene on transport likely results from translocation of the GLUT1 transporter isoform from the cell interior to the surface, via a process dependent upon PI 3-kinase and the cytoskeleton. To assess whether this phenomenon is also involved in the abnormal survival of cells from CML patients, we are currently collaborating with Profs. A.D. Whetton (UMIST) and G. Morgan (Leeds) to develop transport assays sufficiently sensitive for use on clinical samples. A detailed assessment of flow cytometric and other assays using fluorescent sugar analogues has revealed that these lack the necessary sensitivity. However, we have now developed more sensitive radioisotope methods for this purpose. If Bcr-Abl-mediated translocation of GLUT1 is shown to play a role in apoptotic suppression in vivo, this should lead to the identification of novel therapeutic targets for CML.

Molecular profiling of gastric and colorectal cancer - analysis of proteins involved in antigen processing
Dr G.E. Blair, Mr I.G. Martin, Prof. P. Quirke, Mrs A. Trejdosiewicz and Mrs S. Gray

The presentation of tumour-derived antigenic peptides, in association with MHC class I molecules, is an essential prerequisite for cytotoxic T lymphocyte (CTL)-mediated recognition and eradication of neoplastic cells. Strategies that enhance this CTL-mediated tumour "immune surveillance" are a major focus for current immunological research. There is increasing evidence, however, to suggest that a significant proportion of solid tumours evolve "immune escape" mechanisms to evade CTL responses. Our research aimed to evaluate potential immune escape mechanisms in human colorectal tumours. Our results have demonstrated that down-regulation of cell surface MHC class I molecules (the class I heavy chain and the light chain, beta-2-microglobulin) occurs with high frequency at an early stage of colorectal tumorigenesis (p<0.01, Mann Whitney U test). Furthermore, we have identified that reduced expression of the T cell tethering molecules, ICAM-I (intercellular adhesion molecule-I) and LFA-3 (leukocyte function associated antigen-3), occurs in a significant proportion of colorectal adenocarcinomas. The role of the Fas/Fas-ligand system in the tumour-immune dialogue was also being examined. Immune evasion strategies probably arise through natural selection, with tumour cells that can escape CTLs having a profound growth advantage over those that cannot. Our results have important implications for the eventual selection of cancer patients for immunotherapy and give further insights into the dialogue between a malignancy and the immune system.

High risk human papilloma virus oncogenes: Studies on antigen processing and presentation in transformed cells, organotypic cultures and tumours
Dr G. Bottley and Dr G.E. Blair

There is now little doubt that the high risk human papillomaviruses (such as HPV 16 and 18) are closely linked with cervical cancer. Infection of cervical cells with these viruses leads to the expression of the transforming oncogenes E6 and E7 which interfere with the regulation of the cell cycle, and therefore increase the incidence of malignancy.

It is accepted that many tumours, including HPV-associated tumours, display reduced levels of cell surface major histocompatability complex (MHC) class I molecules. It is hypothesised that this results in reduced immune activity against the tumour, and thus enhances tumour growth and survival. The oncogenic adenovirus type 12 (Ad12) is known to down-regulate MHC class I expression at the transcriptional level, via the action of the oncoprotein E1A, and it has also been shown that E7 from HPV 18 also produces transcriptional repression of MHC class I. Therefore it was decided to investigate the physiological action of E6 and E7 from both high risk HPV 18 and low risk HPV 6b. The target cell of HPV in vivo is the skin keratinocyte, and so both HaCaT, a keratinocyte cell line, and primary human keratinocytes were considered to be suitable model systems to investigate.

The initial part of the study examined the changes in MHC class I expression by HaCaT cells following transient transfection of the E7 oncogene. A pilot study revealed the difficulties inherent in a transient system of transfection, including the detection of E7 on a cell-by-cell basis. The HPV 18 E7 gene construct prepared as part of a previous YCR project was ligated into a bicistronic plasmid vector. This vector also directs the expression of a truncated H-2Kk molecule at the cell surface which can be detected by flow cytometry or can be used to purify transfected cells using magnetic beads. It is therefore possible to use two- and three-colour flow cytometry to correlate changes in cell phenotype with E7 expression on a cell-by-cell basis. Use of the magnetic beads allows transfected cells to be analysed by Western blotting, RT-PCR and other techniques independently of non-transfected cells. An additional benefit of using the H-2Kk marker is that the magnetic beads can be used to create a stably transfected cell line in a very short time.

Transient expression of the HPV 18 E7 oncogene in bulk transfected cells has so far resulted in an increase of MHC class I on the transfected cells compared to control cells. This apparently contradicts earlier work performed on transcriptional regulation of the MHC class I pathway, but appears to be due to a soluble mediator released by the transfected cells. Conditioned medium from E7-transfected cells produced an increase in surface MHC class I molecules when transferred to untreated HaCaT cells. We are currently attempting to identify the soluble mediator. An additional observation has been that cells transfected with HPV 18 E7 have an increased incidence of apoptosis and necrosis compared to cells transfected with the control vector. Interestingly, this effect is also seen in cells treated with conditioned medium from E7 transfected cells, so it is possible that this effect is also due to the as yet unknown mediator.

The conclusions that can be drawn from the results so far seem to be that the E7 oncoprotein has an immunostimulatory effect on the transfected cell which results in the release of mediator(s). These in turn seem to upregulate MHC class I and increase apoptosis and necrosis on all cells in the culture, not only those transfected with the oncogene. However, these data have been obtained in bulk transfected cells and need to be extended by studies on clonal cell lines. In addition, comparisons need to be made with low risk HPV 6b E7-transfected cells. Once the identity of the mediator has been confirmed, the system will be utilised to examine primary human keratinocytes in culture and in organotypic culture.

A study of the potential role of the alanine-rich region of oncogenic adenovirus E1A oncoproteins in tumorigenesis
Mrs J. Jarvis and Dr G.E. Blair

The existence of oncogenic serotypes of human adenoviruses has been known since the 1960's, but the molecular determinants of viral oncogenicity have remained unidentified. It is the E1A oncogene of human adenoviruses that determines the oncogenicity of serotypes such as adenovirus 12 (Ad12) and an alanine-rich region, unique to the E1A proteins of Ad12, has been implicated in the oncogenic phenotype of this virus and virally-transformed cells. It is of great interest that alanine-rich regions are also present in transcriptional repressor proteins, suggesting that Ad12-mediated oncogenesis might proceed by a mechanism of repression of key cellular genes involved in tumour suppression. Another possibility is that the alanine-rich region forms a point of interaction between E1A proteins and another, as yet unidentified, protein(s). In this preliminary investigation, we are investigating the latter of these two possible mechanisms using a panel of cell lines we have created where cells have been transformed by either normal, wild-type Ad12 E1A or by Ad12 E1A into which has been introduced an in-frame deletion of the alanine-rich region. Cells transformed by non-oncogenic Ad5 E1A and untransformed cells serve as controls. Proteins interacting with the alanine-rich region of E1A proteins are being studied by co-immunoprecipitation using anti-E1A antibodies. We have also created glutathione-S-transferase-E1A fusion proteins, containing either Ad5 E1A, Ad12 E1A or Ad12 E1A in which the alanine-rich region is deleted. We are using these in protein:protein interaction assays to probe for interactions of cellular proteins with the viral alanine-rich region. Overall, this will provide us with a better view of the likely mechanism involved and its relationship to human cancer.

A mutator mutant of DNA repair polymerase ?: insights into the structural determinants of nucleotide discrimination and replication fidelity
E. Curti, J.B. Sweasy, J. Jäger

DNA polymerase beta (polß) belongs to the nucleotidyl transferase family of polymerases (E.C. 2.7.7.7). Polß is a small DNA repair enzyme (Mr = 39kDa, 340 amino acids) and is involved in base exision repair pathways (BER), specifically mediating the gap-filling synthesis required for the repair of damaged DNA. As DNA lesions such as apurinic and apyrimidinic sites (AP) are believed to occur in eukaryotic cells about 10000 times per cells per day, this repair system is vital for the genomic stability. Mutations in the polB gene have been observed in some forms of human bladder cancer and primary lung cancer, whilst a truncated form of the enzyme has been shown to occur in breast cancers. In collaboration with the Sweasy Lab at Yale Medical School we have identified through a genetic screen a mutant (Phe272->Leu) which shows a marked increase in replication error rates (mutator phenotype). Band shift assays have shown a significant change in the binding affinity for DNA. The aromatic ring systems of Tyr271 and Phe272 have been shown to be important for the discrimination for correct and incorrect dNTP at the ground state.

The crystal structure of this mutant form of polß (Phe272->Leu) has been solved by difference Fourier techniques using data were collected at cryogenic temperature (100K) on a MSC Raxis IV++ image plate detector mounted on a RU300 X-ray generator. The overall completeness is 91% at 2.85Å resolution. Structure refinement and density map calculations were carried out using the program CNS1.0a. The final R-factor is 0.278 for all data between 20 and 2.9Å.

The side chain atoms adjacent to the hydrophobic residue Phe272 show substantial rearrangements compared to the structure of wild type polß. The adaptations in side chains positions are likely a consequence of the decrease in volume of the side chain in position 272. It would appear that this results in an increased volume of the nucleotide binding pocket, whereby the ability of Phe272->Leu polß to recognise and incorporate Watson-Crick base pairs is significantly reduced.

Expression and regulation of the metalloproteinase ECE in prostate cancer
Dr B.A. Usmani and Prof. A.J. Turner

The rate of prostate cancer (PC) in western males is nearly twice that of lung cancer and three times that of colorectal cancer. The endothelin system in prostate cancer is a novel therapeutic target since plasma endothelin (ET) levels are elevated in men with metastatic prostate cancer. This study has focussed on the key enzyme involved in ET synthesis, Endothelin-Converting Enzyme (ECE). Previous immunoblotting studies have revealed that ECE is abundantly expressed in all metastatic cell lines, but weakly expressed in LNCaP cells. In contrast, LNCaP cells were seen to express high levels of neutral endopeptidase (NEP), a closely related metalloproteinase that inactivates endothelin. Metastatic cell lines, however, lacked detectable NEP activity. Primary malignant, cultured epithelial cells lacked ECE expression by immunoblotting analysis, indicating a role for stromal interaction and paracrine production of ECE within the host. It is believed that disturbances in stromal-epithelial interactions in the developing tumour can contribute to prostate cancer progression and metastasis. Reports have shown that growth and invasion can be modulated by vasoactive peptides such as endothelin (ET) and bradykinin. We therefore investigated the effects of ECE and NEP in an invasive prostate cancer cell line in order to elucidate the contribution of these proteinases to the dynamic disturbances in stromal-epithelial interactions during tumour outgrowth.

Using the technique of Matrigel invasion, the effects of stromal cells on the invasive capacity of a PC-3 tumour epithelial cell line was examined. Cell lines used in this study are androgen-sensitive LNCaP, normal transformed prostate epithelial PNT1a, PNT2c2 and P4E6 (kindly provided by Professor Norman Maitland, York), and androgen-independent PC-3 and Du145 cells. Cells were grown either in the presence or absence of ET, bradykinin and ECE/NEP inhibitors. We found that both peptide substrates added exogenously, could stimulate invasion through matrigel by 40-50%. On the other hand, an ECE-specific inhibitor SM-19712, inhibited PC-3 invasion by 50%. Similarly, exogenous NEP inhibited invasion by 50%, an effect that could be recovered by including an NEP-specific inhibitor, thiorphan. These data show, for the first time, a tight correlation between levels of mitogenic peptides available, the enzymes involved in their biosynthesis and degradation, and the degree of invasive ability conferred upon the cell.

Identification of genes that are transcriptionally repressed by Mxi1
Dr S.C.Wright

The development of cancer is often a result of the deregulation of genes that normally act to control cell proliferation and differentiation. Myc proteins are transcriptional activators that promote cell cycle progression and inhibit differentiation; Myc is overexpressed in most human cancers. In contrast, Mad family proteins antagonise the activity of Myc: they inhibit cell division and are associated with growth arrest and differentiation. Mxi1 is a Mad family protein that has been implicated as a tumour suppressor gene in several malignancies including prostate cancer.

In order to elucidate the mechanism of action of Mxi1, it is essential to identify the target genes of transcriptional repression. We have generated cell lines that express inducible alleles of Mxi1 and are currently characterising candidate target genes in conjunction with chromatin immunoprecipitation analysis. This will ultimately benefit the classification and treatment of tumours that are associated with deregulation of the Myc/Mad transcription factor network.

Yorkshire Cancer Research Photodynamic Therapy Group
Centre for Photobiology and Photodynamic Therapy

A multidisciplinary approach to the development of photodynamic therapy for cancer treatment

(A multidisciplinary group of staff from the School of Biochemistry and Molecular Biology, the School of Biomedical Sciences, the Department of Colour Chemistry, the Research School of Medicine, Cookridge Hospital, Leeds General Infirmary, St James's Hospital and Goole and District Hospital.)

Professor S.B. Brown (Principal Investigator) et al

For more than 15 years Yorkshire Cancer Research has provided major support for the development of photodynamic therapy (PDT) by its funding of the Leeds Centre for Photobiology and Photodynamic Therapy. This Centre which is the largest of its kind in Europe and among the global leaders in the development of PDT, now has around 40 scientists and clinicians in the Yorkshire region and many collaborations both in the UK and internationally.

As outlined in previous years, PDT is a developing approach which uses a combination of light and a photosensitising drug (a sensitiser) to selectively destroy unwanted tissue such as tumours. The approach is able to achieve a high degree of targeting partly because of the ability of photosensitisers to concentrate preferentially in tumours relative to normal tissue, but also because of our ability to direct light very accurately to the tumour area.

The past year has seen substantial progress in the development of the Centre's portfolio of photosensitisers towards clinical trials. Our leading drug candidates are now in the final stages of drug development prior to their first use in patients. In total we have screened more than 100 candidate drugs for properties which make them superior to current drugs such as Photofrin and Foscan.

The Centre's drugs consists of families of phthalocyanines and families of phenothiazines. In each case, the central macrocyclic structure largely determines the absorption and sensitising properties and the substituents around the molecule determine the important biological properties. One of the Centre's leading drug candidates (tetra-?-alaninesulphonyl phthalocyanine, or PPA101) has a very strong light absorption around 680 nm which gives a major advantage over Photofrin. One of the drawbacks with Photofrin and Foscan is the prolonged skin photosensitivity suffered by patients following intravenous administration of the drug. Such skin photosensitivity can persist up to three months following drug administration and patients must keep out of sunlight for most of this period, leading to a significant reduction in quality of life. For patients with advanced cancer this is a major barrier to therapy and such a prolonged skin photosensitivity would be completely unacceptable for patients being treated for pre-cancer or early disease. The Centre's drug candidates have been specifically selected to have little or no skin photosensitivity, whilst maintaining strong efficacy against cancers and other lesions. We have carried out extensive skin photosensitivity measurements in two models and in both, PPA101 and our other leading drug candidates show little or no skin photosensitivity under conditions where Photofrin or Foscan have high skin photosensitivity.

Compared with Photofrin, the Centre's drugs are better and stronger sensitisers for tumours. Under conditions where Photofrin induces less than 20% necrosis in experimental tumours, PPA100 induces almost 90% necrosis. It is likely therefore that PPA101 will be effective at much lower doses than Photofrin. Moreover, our data show that maximum necrosis is reached after only a very short drug - light time interval. In patients, we might expect this to be only one to two hours whereas for Photofrin and Foscan, the corresponding period is three to four days. Thus PPA101 would be expected to be much more convenient for clinical use since the light delivery could be given in the same treatment session as the drug.

Perhaps the greatest advantage of PPA101 over existing drugs is the selectivity achieved between tumour and normal tissue. Following administration of PPA101, when light is delivered through the skin to a subcutaneous tumour, there is complete tumour necrosis but little or no damage to overlying skin or underlying muscle. Again, these results have proved much superior to those obtained by administering Photofrin under the same conditions.

Progress has also been made on the synthesis of a new family of phthalocyanine sensitisers which it is hoped will have the same beneficial properties as the PPA101 family, but will be easier to manufacture and purify.

These achievements in drug development have now been consolidated in five patent applications to protect the relevant intellectual property. Whilst this has meant a delay in the open publication of the data, it is anticipated that several papers resulting from the work will be published within the next year.

It is anticipated that these drugs will be most suited to the treatment of early cancer and pre-cancerous conditions. With current increases in screening methods and projected further demand for early screening, there will be a greater and greater need for relatively mild treatments to treat either early cancer or to give preventative treatment to prevent pre-cancerous lesions progressing to cancer. Examples here are the treatment of Barrett's oesophagus which can lead to cancer of the oesophagus and CIN which can lead to cancer of the uterus.

A company, Photopharmica, has been formed in order to progress these developments commercially. With active involvement of YCR, it is hoped that this company will provide the vehicle by which the major investment of YCR and the University in PDT can be harnessed to benefit large numbers of patients.

YCR Nurse support for Cancer Clinical Trials within Yorkshire

Dr S A Mason - Head of Unit (Joint)
Ms V Napp - Senior Trial Co-ordinator

Four whole-time equivalent research nurses (three full-time and two part-time) were funded by the YCR from 1997, with the last contract terminating in February 2002. They have worked in the following hospitals throughout Yorkshire:

Castle Hill Hospital, Hull
Clayton Hospital, Wakefield
Cookridge Hospital, Leeds
Dewsbury District Hospital
Halifax Royal Infirmary
Harrogate District Hospital
Huddersfield Royal Infirmary
Pinderfields Hospital, Wakefield
Pontefract General Hospital
Princess Royal Hospital, Hull
St James's University Hospital, Leeds

Hospitals were chosen by a small scientific review panel as a result of competitive bidding by individual centres. The NYCTRU provided training and co-ordination support to the nurses. Management of the nurses was provided locally by a Research Nurse Manager. Non-commercial trials mainly in the area of breast, colorectal and lung cancer were identified as those which would benefit from research nurse support.

The table below outlines the significant improvement in both the number of active trials and the number of patients entered into these trials following the employment of the YCR nurses.

YCR nurses employed