Cancer Medicine and Pathology, Cancer Research UK Cancer Medicine Research Division

Urological Cancer Team (R Banks, P Harnden and P Selby)
The focus of this team is on clinical and laboratory studies in patients with urological cancer, based primarily around immunotherapy and the identification of novel therapeutic targets and tumour markers in patients with renal cancer and the use of differential display expression analyses and cDNA microarrays to identify molecular pathways of bladder tumour development.

Proteomics-based studies, identifying new renal cell carcinoma antigens and markers, have initially centred around 2-D PAGE with computerised analysis of gel images and subsequent identification of proteins of interest by mass spectrometry (in collaboration with Dr Nick Totty, Cancer Research UK Protein Sequencing Laboratory) and are now well established. Initial comparisons of lysates from normal and malignant tissue have found 32 proteins to have an increased expression in at least four out of six grade 3 RCC samples and 41 proteins to be down-regulated. Ten of the differentially expressed proteins identified this far are part of the glycolytic pathway, presumably reflecting the increased energy requirements, and many are regulated by HIF-1. The majority of these proteins, including annexins, manganese superoxide dismutase, vimentin and elongation factor 2 have previously been reported to be up-regulated in other tumour types. Two forms of lactate dehydrogenase-M, triosephosphate isomerase and phosphoglycerate kinase have been found and the significance of these is currently being investigated. Several of these proteins are now being examined immunohistochemically in a small number of samples. Any putative markers will then be evaluated using our sample bank which now contains samples of tissue, urine and blood from approximately 140 patients with renal cell carcinoma, and interpreted in the light of the clinical data. Alternative strategies to enhance the detection of lower abundance proteins using large format 2D-PAGE are also being evaluated, including the use of zoom IPG strips and sub-cellular fractionation.

One of the problems associated with using tissue lysates is of course sample heterogeneity in terms of the multitude of cells present. The possibility of using laser capture microdissection or of preparing short-term primary cultures from the normal and malignant renal tissue has been explored as alternative approaches. After an extensive evaluation, although LCM is useful for preparing small amounts of tissue for use with western blotting or alternative proteomic techniques such as SELDI (Surface Enhanced Laser Desorption Ionisation), the time-consuming nature and the large amount of protein required for large format 2-D gels precludes its routine use in such studies. This is to a certain extent tissue-dependent but will always limit analysis to the most abundant proteins. Using short term primary cultures, a range of proteins have now been identified which are expressed at either higher or lower levels in lines derived from malignant tissue compared with normal kidney and these are currently awaiting identification.

Using the SPEAR approach, a novel modification of the SEREX where 2-D western blots are probed by autologous serum, several proteins have been identified as being recognised by patient's serum. One of these is thymidine phosphorylase (platelet derived endothelial cell growth factor) which has been previously shown to be over-expressed in RCC. This is now being investigated further in in vitro studies initially, as a possible immunotherapeutic target.

Studies using SELDI to profile urine samples from patients with RCC have now shown reproducible differences between tumour and normal urines with several proteins appearing to be specific enough to warrant purification and identification as potential markers. This work is ongoing. Following earlier work with cDNA microarrays, the relevance of a possible FGF autocrine loop in the pathogenesis of RCC is being investigated with the principle involvement of FGF8b. A novel finding of an over-expression of BIGH3 from both cDNA microarray and suppression subtractive hybridisation work is currently being followed up in collaboration with Bristol Myers Squibb.

To isolate genes involved in bladder tumour development, we compared early neoplastic lesions with normal urothelium, obtaining specific cell populations by laser capture microdissection. We generated suppressive subtractive cDNA libraries and identified differentially expressed genes for further investigation using immunocytochemistry, microarrays and urine testing.

The team continues to carry out clinical research into biological and chemotherapy for renal, bladder and prostatic cancer. Trials active this year have included: mycobacterial vaccine, thalidomide therapy and single and combination cytokine therapy for renal cancer; combination chemotherapy for fit patients with advanced bladder cancer and bladder cancer following apparently complete surgery; chemotherapy for prostatic cancer. Molecular diagnostic techniques for prostatic cancer based on real time RT-PCR appear to be solving some of the technological problems surrounding conventional RT-PCR and to be superior to alternative nucleic acid amplification techniques and to microsatellite based methods of detecting tumour related DNA.


Lymphoma, Myeloma, Sarcoma
Our international clinical trial in multiple myeloma is now concluded and is the largest trial of high dose chemotherapy with peripheral blood stem cell transplantation in the world (in collaboration with Professor Gareth Morgan and Dr Tony Child of the Academic Unit of Oncology & Haematology). Novel approaches to the measurement of quality of life in patients in this trial have been developed and a comparison of the quality of life achieved with the intensive therapy has also been carried out. A broad portfolio of lymphoma clinical trials includes a novel combination chemotherapy for advanced and relapsed Hodgkin's disease (the GEMDCOPP regimen) and a contribution made by our lymphoma clinical practice to the valuation of monoclonal antibody therapies, chemotherapy combinations and high dose chemotherapy with stem cell transplantation. Ken MacLennan and Reuben Tooze (NHS Trust) with the British National Lymphoma Investigation, the European Organisation for Research and Treatment of Cancer and the United Kingdom Children's Cancer Study Group, continues to analyse lymphoma classification and the prognostic significance of histological sub-types of non-Hodgkin lymphoma and Hodgkin's disease. Pilot experiments to explore molecular genetics of putative Reed-Sternberg cell precursors and the molecular mechanisms underlying cell morphology in B cell lymphoma/leukaemias are being conducted.

Service re-organisation in West Yorkshire has progressed this year with a commensurate rise in the number of referrals to the sarcoma team. This has stimulated our contribution to the EORTC soft tissue and bone sarcoma group clinical trials. New clinical trials include a phase III trial of the new protein tyrosine kinase inhibitor STI571 (Glivec) for GI stromal tumours.

Further experience has been gained in the assessment of translocation fusion products by TaqMan assay (Dr Will Merchant) and how to factor this into the clinical management of our patients.

Sarcoma is an attractive field in which to expand our laboratory research program. Unusual for solid tumours, a number of characteristic chromosomal rearrangements have been described and a small laboratory interest group with the Academic Unit of Pathology is developing PCR based assays to detect translocation fusion products to augment traditional histopathological diagnostic techniques.


Melanoma
The Melanoma Unit continues to develop approaches to the immunotherapy of melanoma. In the clinic, recruitment continues into EORTC collaborative group studies of adjuvant pegylated Interferon in stage III melanoma and chemoimmunotherapy for stage IV melanoma. A phase I/II study of MAGE-3 protein vaccination in collaboration with the Ludwig Institute is under way and a phase I study of DNA vaccination with polyepitope DNA vaccine encoding a string of melanoma T-cell epitopes has been developed in collaboration with Applied Immunology Laboratory. This is being submitted to the Gene Therapy Advisory Committee.

In vitro experiments investigating the interaction between tumour cells and dendritic cells, are being conducted by Dr Dearbhaile O'Donnell, Cancer Research UK Clinician Scientist. We are investigating the factors that affect tumour antigen processing and their affect on dendritic cell interaction with T-lymphocytes. Using an embryonic stem cell system, we have generated functional dendritic cells and have established the conditions for this. This system will be used to study candidate promoters for targeting transgene expression and will allow studies of DC lineage. In collaboration with the National Blood Service, we have established funding to build and equip a clean laboratory facility to allow dendritic cell vaccinations for human studies.

Dr Jackson, Cancer Research UK Clinician Scientist, is studying the mechanisms of interferon resistance in malignant melanoma. Using the DNA dense arrays to compare gene expression of interferon sensitive and resistant lines, we hope to identify candidate genes involved in interferon resistance.

In conjunction with the Genetic Epidemiology Division, work continues directed towards understanding the genes predisposing to melanoma, and their interaction with the environment. In families at high risk of melanoma, mutations in CDKN2A are known to underlie susceptibility in a proportion of families but all groups internationally have failed to identify mutations in a percentage of families linked to chromosome 9. Dr Mark Harland has now however identified deep intronic mutations in six UK families for the first time and is screening for other such mutations in conjunction with the Mutation Detection Facility. The Leeds group works with the Melanoma Genetics Consortium, which Julia Newton Bishop continues to chair. We were successful in funding this consortium via the US National Institute of Health, this year, which will allow pooling of data from the US, Australia and seven European groups over the next five years.

The group is extending its area of interest towards understanding the genes which control relapse from melanoma, and the effect of the environment. A large cohort study of melanoma has therefore begun recruiting in December 2000, which will accrue for the next five years. A nested case control study of late relapsing melanoma is running concurrently which will accrue till 2003.


Gastrointestinal Tract Cancer (MT Seymour, A Anthoney and A Melcher)
Projects in colorectal molecular pathology are run collaboratively with Prof Phil Quirke (University of Leeds). The project in intraperitoneal therapy is run collaboratively with Mr Paul Finan (University of Leeds).

Our work on using microsatellite analysis to select patients for chemotherapy was reported last year.

We are now testing these findings prospectively with an expansion of the number of patients and of the number of DNA microsatellites on 17p and 18q. We have obtained normal and tumour tissue from a further 1400 patients in prospective randomised trials of "+/- adjuvant chemotherapy", and have extended our DNA microsatellite analysis to include several additional markers on 18q and 17p and others on 14p, 12p and 2p. A robotic system of DNA processing is making it possible to process the large numbers of samples. We are now creating tissue arrays from the same large clinical series to allow rapid immunohistochemistry for cancer- or treatment-associated proteins. In collaborations with Genetech, Affymetrix and Glaxo-Wellcome we are measuring expression of a wide range of genes in subsets of the clinical trial material to guide further focused studies in the full population. As the project develops we hope to make use of the facilities generated through the ICRF expression profiling and microarray facility at Hinxton Hall.

When trying to activate an immune response against a tumour, the way in which tumour cells die may be critical to successful immune priming. Our previous work has shown, in animal models, that necrotic cell death is effective at loading dendritic cells with tumour-associated antigens for presentation to T cells, whereas apoptotic cell death is not. We are now continuing and extending these studies in preclinical and clinical studies focused on colorectal cancer. In the laboratory, necrotic and apoptotic tumour cell death can be controlled using chemotherapy and radiation, and compared for immunogenicity. This has specific relevance to the tumour cell death currently triggered by chemo/radiotherapy in patients, and whether this can be harnessed for immunotherapy. We are collecting clinical samples from patients treated with short course pre-operative radiotherapy to analyse for apoptotic/necrotic tumour cell death and induction of potential immunogenic signals such as heat shock proteins. Further laboratory projects are focused on allogeneic tumour cell vaccines for colorectal and other cancers, and the cellular and molecular interaction between dendritic cells and tumour cells, and its importance in the control of the anti-tumour immune response.

We are involved in the clinical development of several new drugs with differing mechanisms of action. These include the conventional cytotoxic drugs oxaliplatin, mitomycin and irinotecan, a farnesyl transferase inhibitor (R115777) and immunotherapies based on 17-1A (Edrocolomab) and a gastrin-diphtheria toxin conjugate immunogen (G17DT). In particular, we have been involved in the development and running of the current MRC CR08 (FOCUS) trial in advanced colorectal cancer, which in includes randomisation to 5FU combinations with oxaliplatin or irinotecan. The same approaches that we have used to address benefit from 5FU in the adjuvant setting are now being extended to these clinical trials, where the molecular correlates of benefit may be different. This work is being supported by a Cancer Research UK Bobby Moore research fellowship, Dr Julian Adlard.


Women's Cancer (T Perren, G Hall, A Hanby)
The team specialises in the treatment of women with breast cancer or ovarian cancer and is closely integrated into the multi-disciplinary teams of the Leeds Cancer Centre. Our aim is to deliver high quality and appropriate treatment within the context of a clinical trial where possible. Over the past 12 months around 50% or our breast cancer patients have entered clinical trials as have approximately 70% of our ovarian cancer patients.

We have now completed and analysed our study on the maintenance effect of interferon alpha in patients with ovarian cancer who have achieved at least disease stabilisation following first line chemotherapy. The analysis does not show any improvement in survival for patients receiving interferon. Although with 300 patients randomised the statistical power is still limited, it is the largest trial addressing this question that has been completed internationally.

During the course of this year we have recruited into the following first line chemotherapy trials: the SCOTROC trial comparing Carboplatin and Paclitaxel with Carboplatin and Docetaxel which has now closed. In conjunction with Dr Ganesan from the Cancer Research UK Unit in Oxford we are recruiting patients into a trial of Carboplatin alone versus Carboplatin plus the anti-angiogenesis agent Thalidomide. Open to recruitment is OVCAT and EBMT study of standard dose carboplatin and taxol versus high dose sequential chemotherapy. We have recently begun recruiting in collaboration with Dr Gore and Professor Kaye at the Royal Marsden into a phase II feasibility study of sequential Carboplatin/Taxol and Gemcitabine as part of the SCOTROC 2 programme. In advanced ovarian cancer we have continued recruitment into ICON4 and MRC study of carboplatin versus carboplatin plus taxol in platinum sensitive advanced ovarian cancer. In the lab we are contributing blood samples to the collaboration coordination by Frannie Balkwill to examine TNF polymorphisms in patients with ovarian carcinoma. An interesting project is also under way looking at comparative genomic hybridisation in clear cell ovarian carcinomas together with genetic and immunohistochemical analysis. More recently, in collaboration with Dr R Banks, a proteomics-based approach has been adopted to search for novel ovarian cancer markers or targets. We have recently appointed Dr Geoff Hall as Senior Lecturer to the team with a remit to develop or translational research programme.

In breast cancer we have continued to contribute patients to a broad range of national randomised studies. The Unit continues to be represented on the Steering Committee of the ABC, NEAT and ATOM trials and is now represented on those for TACT and TANGO. During the course of the year we have developed a sequel to our local phase I/II dose escalation study of the novel combination of Epirubicin, Taxotere and continuous infusion of 5FU in patients with metastatic breast carcinoma. The new trial will evaluate the combination of weekly taxotere with capecitabine and epirubicin. This trial will also be carried out in conjunction with colleagues in the Yorkshire Breast Cancer Research Group. Recruitment into the trial looking at the novel combination of Vinorelbine and Carboplatin in patients with metastatic breast carcinoma has been suspended to recruitment due to higher than expected toxicity. The double-blind randomised placebo controlled trial of Theratope vaccine in patients with metastatic breast carcinoma who have obtained at least stabilisation of their disease with first line chemotherapy has been completed, but may patients remain on follow up. We continue to recruit to the Anglo-Celtic Collaborative Group II study part A (Adriamycin plus Taxotere versus Adriamycin plus Cyclophosphamide as primary medical therapy in potentially operable breast cancer) as well as to IBDIS I (an international collaborative group study), a trial of high dose chemotherapy for metastatic breast cancer.

We have recently recruited Professor Hanby from the Breast Unit and together with Dr Val Speirs we are planning to strengthen our translational research activity. Dr Speirs is currently active in the investigation of Erβ in breast cancer as a predictor of response and prognosis. Professor Andrew Hanby moved to Leeds from London, where he had been joint head of the ICRF Hedley Atkins Breast Pathology Laboratory. Though employed by Leeds Medical School he is an Honorary Cancer Research UK member and plans to integrate his efforts with those of Cancer Research UK in Leeds and also with Cancer Research UK Units elsewhere. Urgent issues being addressed, ultimately beneficial to the Unit, include the establishment of a breast pathology database which can be integrated into existing oncology and BASO databases and the introduction of consent for the use of reserve surgically excised tissues in breast cancer research. He continues his work on the taxonomy of breast cancers in collaboration with Drs Tomlinson and Roylance in the Molecular and Population Genetics Laboratory at Lincoln's Inn Fields.


Cancer Care Epidemiology (D Forman)
Collaboration continues in work on cancer care epidemiology with the Northern and Yorkshire Cancer Registry and Information and Service (Professor David Forman and Professor Bob Haward). This has been mainly concerned with studies of breast cancer management, investigating two issues that can not be addressed by randomised trials. Firstly we have been looking at whether workload volume (surgeon caseload) has an effect on survival. This extends work published 6-7 years ago showing breast cancer patients had an improved outcome if managed by surgeons with a high annual caseload. Since that time, there has been a further 'concentration' of breast cancer care within the UK and the question is whether the same volume effect persists. We analysed over 16000 breast cancer patients, diagnosed in Yorkshire between 1986 and 1994. Of these, 6% were managed by surgeons with an annual caseload of less than 10 cases, 26% by surgeons with 10-29 cases, 33% with 30-49 cases and 35% with 50 or more per annum. Overall five-year survival was 62% with a statistically significant difference in outcome between patients managed by low and high volume surgeons (58% and 67% survival in the lowest and highest workload groups respectively). This variation can not be explained by differences in case-mix.

We have also been looking at delays experienced by cancer patients prior to receiving treatment and the effect this has on survival. There is variability in the extent of delay according to the type of cancer and as a result of hospital practices and procedures. Following work on the delays experienced by breast cancer patients prior to initial treatment, (in which we showed that delays of three months do not seem to be associated with decreased survival), we are now undertaking a detailed study of the time interval between receiving surgery and starting radiotherapy. The aim of radiotherapy after surgery for early breast cancer is to reduce the risk of local recurrence. This is particularly important for patients who have received breast conserving surgery, the rates of which have been increasing in recent years. Demands on radiotherapy facilities are, therefore, increasing and this is leading to longer waiting times. We have analysed data on over 12000 breast cancer patients diagnosed between 1986 and 1998 who have been treated with both surgery and subsequent radiotherapy. We are investigating the impact of clinical, socio-economic and access factors on the delay interval and then looking at whether there is any impact of delay on survival.


For a list of refereed research papers, see Publications (in navigation on left).