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Manchester Cancer Research Centre

Research Report 2009/10

Contents

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Chair’s Foreword 4

Introduction 5

The MCRC Partnership 9

Manchester Cancer Research Centre 10Conference - Harnessing Apoptosis

Chris Morrow, Kathryn Simpson, Luke Harrison,

Tetyana Klymenko, Tom Owens and Fiona Foster

Lung Cancer Circulating Tumour Cells as 16

Biomarkers and a Window to Metastasis Biology Tim Ward, Jian-Mei Hou, Matthew Krebs,

Fiona Blackhall and Caroline Dive

DNA Damage Response 20Ivan Ahel

Drug Discovery in the Manchester 24

Cancer Research Centre

Donald Ogilvie and Allan Jordan

Genetic Medicine and the 28

Genesis Prevention CentreD Gareth Evans and Anthony Howell

Cancer Genomics in the Era 34

of High Throughput SequencingCrispin Miller, Jenny Varley and Stuart Pepper

Imaging Research at the Manchester 40Cancer Research Centre

Alan Jackson and Kaye Williams

Obesity and Cancer 46Andrew G Renehan

Role of MKK4 in Ras-Induced Cancers 52Cathy Tournier

The New Patient Treatment Centre 56Malcolm Ranson

Author Biographies 60

Manchester Cancer Research Centre Research Report 2009/10

Manchester Cancer Research CentreThe University of ManchesterWilmslow RoadManchester M20 4BXTel: +44 (0) 161 446 3156www.manchester.ac.uk/mcrc

Founding Partners:

The University of ManchesterOxford Road Manchester M13 9PL Tel: +44 (0) 161 306 6000www.manchester.ac.uk

The Christie NHS Foundation TrustWilmslow RoadManchesterM20 4BX Tel: 0845 226 3000www.christie.nhs.uk

Cancer Research UKAngel Building407 St John StreetLondonEC1V 4ADTel: +44 (0) 20 7242 0200www.cancerresearchuk.org

Copyright © 2010 Manchester Cancer Research Centre

54

Welcome to the 2009/10 Research Report of the Manchester Cancer Research

Centre (MCRC).

The Research Report provides a glimpse into some of the challenges and

achievements over the last two years and emphasises the importance of the

research being undertaken at the MCRC. As a non-scientist and a non-

clinician, what strikes me about the work highlighted within the report is the

diversity of the research undertaken and the innovative approaches being

used to address some of the key outstanding issues in our understanding, and

therefore management, of cancer. This diversity is indicative of the complexity

of cancer as a disease and the need for multifaceted approaches in order to

drive research that leads to improvements in how patients are diagnosed and

treated and improvements in patient outcome.

For the MCRC, 2009/10 has clearly been a successful year, with the fruition of

several long-term strategic initiatives such as the establishment of the MCRC

Drug Discovery Centre and the physical initiation of the new Patient

Treatment Centre at The Christie. These developments provide tangible

evidence of the progress that has been made at the MCRC but also symbolise

the other less visible achievements being made through improving our

understanding of basic biological mechanisms in cancer in order to develop

novel tools and treatment strategies for cancer patients. Importantly, the

research base has been strengthened by attracting leading international

researchers to join the MCRC, giving fresh impetus to research programmes,

adding to the expertise available and bringing opportunities for beneficial

collaborations and the development of holistic research initiatives. This fertile

and nourishing environment is one which fosters and nurtures progress.

Outside of the clinical and scientific world, challenges in the environment we

work in drive innovation and can be motivational; the same is true within

cancer research. The scope and breadth of MCRC research mirrors the

challenge of cancer and highlights the MCRC’s commitment to embrace this

challenge. Ambitious but achievable plans are intended for 2011 and beyond

– in the next Research Report we hope to be able to share with you how some

of these key objectives have been met.

Dr Michael OglesbyChairManchester Cancer Research Centre

Chair’s Foreword

The remit of the MCRC covers the spectrum from basic research through to

translational and clinical research. Our aim is to elucidate the cellular processes

and mechanisms that drive the development and establishment of cancer in

order to identify opportunities for therapeutic intervention and to evaluate

these interventions in clinical trials. This report highlights some key research

areas that exemplify the advantages of this rational and strategic bench to

bedside approach.

Cells have a remarkable capacity to repair DNA damage, a capacity which is

crucial in ensuring accurate replication of the cell’s genomic material.

Aberrant DNA repair mechanisms lead to accumulation of genetic defects

causing disease and the ability to circumvent normal repair mechanisms is

characteristic of many forms of cancer. In order to allow accumulation of

genetic defects, cancer cells may sacrifice specific DNA repair pathways,

making them uniquely susceptible to molecules that impair or inhibit specific

DNA damage response routes. The report by Ivan Ahel, a new recruit to the

MCRC, highlights research being undertaken to better understand the

molecular mechanisms and players in DNA damage response in order to

exploit cancer cell defects in the maintenance of genomic integrity. This

research has led to the identification of novel molecular mediators in DNA

repair processes. Studies on DNA repair mechanisms are particularly exciting

right now since recently developed inhibitors of certain repair pathways are

showing great promise in the clinic.

Despite efficient repair mechanisms, genomic mutations that drive

tumourigenesis do occur leading to cancer development. A major challenge in

maximising the effect of therapeutic interventions is patient stratification

and the identification of those patients whose tumours have a genetic pattern

that makes them more likely to respond to a particular therapy. Since the

publication of the human genome a decade ago spectacular advances in DNA

sequencing technology and techniques have been made and these advances

Introduction

Dr Michael OglesbyChairManchester Cancer Research Centre

Professor Nic JonesDirectorManchester Cancer Research Centre

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provide great promise for defining the genetic landscape of individual tumours. The report by Miller et al

details the potential and promise of high throughput cancer genomics and the advanced DNA analysis

technology and how they are being used by MCRC researchers to better understand tumour initiation,

progression, prognosis and response to therapies.

The power of defining the genome landscape to match patients to particular treatments depends on

thorough biological knowledge and recognition of the exact mutations that drive tumour growth. A major

focus of MCRC research is to develop gene signature profiles for a range of cancers, the most well

understood currently being breast cancer. The report from Evans and Howell describes the contribution

MCRC researchers have made to a more detailed understanding of breast cancer through rigorous genetic

analysis of samples from thousands of patients. A key goal is identifying genetic profiles that predispose

individuals to tumour development and to use this knowledge to apply preventative interventions to

identified high-risk target populations. MCRC researchers are part of a national initiative that aims to

optimise existing screening programmes by refining risk assessment models and identifying novel

additional risk factors that can improve risk prediction. MCRC researchers were the first to conduct a

comprehensive genetic study on neurofibromatosis type 2, a malignancy that affects skin and nervous

tissue. Based on over a decade of experience, they have secured funding for a national programme on the

management of this disease, a programme which aims to improve patient outcome by identifying and

promoting best practice and a consistent approach to patient management.

Within the MCRC there are substantial efforts to characterise and validate biomarkers that can measure or

predict therapeutic response. A particularly exciting and important area of research involves the

characterisation of circulating tumour cells (CTCs) and evaluating the potential of these cells to act as

biomarkers: to ask whether changes in CTCs can provide prognostic information or an early indication of

therapeutic response, Given the difficulty of accessing biopsy material from certain tumours for genomic

analysis and other studies, for example because of their location, the availability of serum-borne material

that can be routinely monitored before, during and after therapy has significant advantages in terms of

convenience and logistics for both patients and researchers. This approach is described in the report from

Ward et al, and evidence provided for encouraging early results in lung cancer.

The study of molecules as potential biomarkers is not the only method of measuring response to therapy:

advanced imaging approaches are also showing great promise in this regard. The newly formed MCRC

Imaging Group is tasked with promoting the integration of advanced imaging technology across the

breadth of MCRC research in order to fully exploit the range of expertise and world-class facilities available

within the MCRC partnership. Detailed within the report from Jackson and Williams is research aimed at

identification and validation of imaging biomarkers which are directly relevant to tumour behaviour or able

to monitor the impact of therapeutic interventions, preclinical imaging to investigate cell proliferation,

metabolism and cell death, and the use of imaging within clinical research programmes using

multimodality imaging techniques across a range of cancer types.

Understanding and predicting patient response

to therapy including adverse treatment

responses is not limited to studies of genetic

factors but also encompasses research into the

impact of lifestyle and dietary habits on

outcomes. With emerging evidence for obesity

as an adverse prognostic factor in cancer and a

potential marker for increased adverse

treatment effects, MCRC researchers are

conducting studies to better understand and

unravel the complex relationship between

cancer and obesity using both in vitro and in vivo

models, as described in the report from Renehan.

Following groundbreaking research into the

epidemiology of excess weight and cancer,

researchers are now exploring its impact on

treatment response and outcome, the possible

role of molecular mediators such as insulin on

cancer risk and progression and developing

robust statistical methodology for the

evaluation of potential biomarkers in clinical

trials.

98

The University of Manchester

Over the last five years, the partnership has established the MCRC as one of the

largest research centres of its kind in Europe and has strengthened its capability

across the full spectrum of cancer research activity. The MCRC is continuing to

recruit eminent researchers to lead its basic, translational and clinical research

portfolio. The real strength of the MCRC is evident in the closer working relationships

with the partner NHS Trusts. Building on these relationships is vital to expanding our

basic research effort and fostering the translation of the knowledge we gain in to

better patient care.

Professor Dame Nancy Rothwell Professor Rod Coombs

President & Vice-Chancellor Deputy President & Deputy Vice-Chancellor

The Christie NHS Foundation Trust

The MCRC partnership has maximised the impact of activity in cancer research and

is making Manchester on of the largest and most significant centres for cancer

research and treatment.

We are driving forward ambitious plans to provide world class services to our

patients, and developing our clinical research is an important aspect of this. The

new Patient Treatment Centre, which will be the largest early phase clinical trials

unit in the world, provides a fantastic example of the benefits of working in

partnership and brings huge benefits to patients, which is ultimately what our

efforts are all about.

Lord Keith Bradley Caroline Shaw

Chairman Chief Executive

Cancer Research UK

We are proud to have a long association with cancer research in Manchester, and

the MCRC partnership fits exemplifies our vision that ‘Together we will beat cancer’.

Bringing scientists and clinicians closer together will help us to improve our

understanding of cancer and find out how to prevent, diagnose and treat the many

different kinds of the disease. We are committed to building strong partnerships to

maximise the opportunities for discoveries in the laboratory to translate into

benefits for patients. The MCRC is a great example of such a partnership and, with

the development of the Drug Discovery Centre, we have the potential to yield new

treatment options that can have a real impact on cancer patients.

Michael Pragnell Harpal Kumar

Chairman Chief Executive

The MCRC Partnership

Cancer development and progression is a complex process and so also are the mechanisms that control

this process. Cell signalling pathways provide a mechanism by which cells are able to respond and adapt to

changes or stimuli in their environment and play an important role in controlling cell behaviour. Activation

of specific pathways triggers a cascade of events and reactions ‘downstream’ from the initiating activation

point which subsequently impact cell behaviour. Research by Cathy Tournier and colleagues within the

Molecular Cancer Group has provided the first genetic evidence that signalling downstream of MKK-4, a

molecule with potent cell signalling properties, plays an essential role in the formation of skin tumours.

The findings which are featured in this report are particularly noteworthy as until now there has been

conflicting data on the role of MKK-4 in tumour development and therefore its potential as a possible target

for anticancer drug therapy. The team have now been awarded a grant from Cancer Research UK to elucidate

the role of MKK-4 in tumour initiation and progression, under normal conditions and in response to

anticancer therapies.

Improving patient outcome requires innovative approaches to treatment, including the use of existing drugs

in more effective ways such as novel combination strategies and the identification of new target

populations. In parallel, the identification and development of new drugs remains crucially important and

the report from Ogilvie and Jordan focuses on the MCRC Drug Discovery Centre, which is part of a

coordinated policy within Cancer Research UK to increase and prioritise research in this area. In 2009 the

new Centre became a reality with the skills, facilities and partnerships now in place to drive the

development of novel small-molecule based therapies.

Key to our success in improving patient treatment is the ability to thoroughly evaluate promising new

agents in early phase clinical trials. The new £35 million Patient Treatment Centre at The Christie opened in

November 2010, and is described in the report from Ranson. The new Centre will provide for a doubling of

clinical trials activity with an emphasis on increasing capacity for early phase clinical trials. In addition, the

Centre allows for an optimised patient care pathway by bringing together core facilities such as service

chemotherapy and pharmacy under one roof. The expansion and continued commitment to clinical trial

research enhances the international reputation of The Christie and MCRC partners in the conduct of high

quality clinical research with the ultimate goal of improving outcomes for patients with cancer.

We are in a hugely exciting era in cancer research with unrivalled opportunities in terms of increased

knowledge, sophisticated technology and dedicated resources. A recurring theme throughout the highlights

featured in this report is that the challenge to develop a personalised and individual approach to treatment

based on genetic and molecular profiles of individual patients and their disease is being embraced. We aim

to build on the progress already made and to use the advantages that partnership and collaboration within

the MCRC brings to deliver benefits to individuals with cancer.

Professor Nic Jones

Director

Manchester Cancer Research Centre

The Manchester Cancer Research Centre was founded by The University of Manchester, The Christie NHS

Foundation Trust and Cancer Research UK and, with the advent of the Manchester Academic Health Science

Centre (MAHSC), has been expanded to include Central Manchester University Hospitals NHS Foundation

Trust, Salford Royal NHS Foundation Trust, University Hospital of South Manchester NHS Foundation Trust,

Manchester Mental Health and Social Care Trust and NHS Salford (Salford Primary Care Trust).

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therapeutic targeting of IAPs in cancer cells induce apoptosis. His IAP

antagonists caused Ub-mediated degradation of c-IAP1 and 2, lNF-κB

activation and up-regulation of TNFα. The resulting autocrine/paracrine TNFα

signals induced TNF-RI-dependent apoptosis. In contrast, XIAP neutralisation

by IAP antagonists synergised with anti-DR5 or FasL to induce TNFα

independent apoptosis bypassing the requirement to engage the

mitochondrial apoptosis pathway. Thus, IAP antagonists invoke multiple hits

on apoptotic signals in cancer cells.

Anthony Letai (Dana Faber Cancer Research Institute, Boston) discussed the

molecular mechanisms that set a threshold for drug-induced apoptosis to

explain cancer cell sensitivity and resistance. He described three mechanisms

by which cancers evade pro-apoptotic signals emanating from tumour

microenvironment or from oncogene activation by altering Bcl-2 family

member levels. Significantly, if cancers have evolved to over-express anti-

apoptotic Bcl-2 family proteins they are ‘primed’ for apoptosis and should

readily engage apoptosis after BH-3 mimetic drug treatment. Tony described

his technique, BH-3 profiling, which determines whether a cancer cell is

‘primed’; an approach showing some potential to predict drug response in

lymphoma patients. Saul Rosenberg (Abbott Laboratories, Illinois) described

the development of BH-3 mimetics. ABT-737 was developed using NMR–based

chemical library screening linked to structure-based design, to identify small

molecules that bind with sub-nanomolar affinity to BCL-2, BCL-xL, and BCL-w.

In preclinical studies ABT-737 exhibited single agent activity in almost all

tumour types, but enhanced cytotoxicity of chemotherapy and irradiation

across a wider cancer cell line panel. The orally bioavailable clinical candidate,

ABT-263, is now in phase I/II clinical trials with some early promising results.

The focus switched to clinical utility of drugs targeting direct regulators of

apoptosis and techniques to measure apoptosis in cancer patients. Malcolm

The Manchester Cancer Research Centre Conference: Harnessing Apoptosis

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Manchester Cancer Research Centre Research Report

The inaugural Manchester Cancer Research Centre Conference entitled

‘Harnessing Apoptosis’ brought together scientists and clinicians from across

Manchester and around the globe to listen to presentations from

international leaders in this field. The conference, which was held at The

Palace Hotel on 17-20 January 2010 attracted 120 delegates, was organised by

Caroline Dive (Paterson Institute for Cancer Research), Charles Streuli

(Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, The

University of Manchester) and Esther Walker (MCRC Operations Manager).

Following a warm welcome to Manchester from MCRC Director Nic Jones, the

opening plenary lecture by Doug Green (St. Jude’s, Memphis) gave an

introduction to apoptosis signalling pathways and focused on reconciling an

ongoing controversy regarding two proposed models of regulation of

mitochondrial outer membrane permeabilisation (MOMP) by Bcl-2 family of

proteins. His lab used a BH3-domain-graft method to take advantage of the

differing affinities of Bcl-2 family proteins for each other, to dissect whether

regulation of MOMP occurred by the ‘direct activation/de-repression’ or

‘neutralisation’ model. His new data suggested that both models can occur

dependent upon cellular stress levels. An important ramification may be that

cells are sensitised to death induced by Bcl-2 targeted drugs (BH-3 mimetics)

when ‘primed’ by low levels of cellular stress.

The conference continued with sessions on Inhibitors of Apoptosis Proteins

(IAPs) and the Bcl-2 family. Data presented by Pascal Meier (Institute of Cancer

Research, London), provided further insight into IAP function in vivo. His

group identified a novel mechanism by which IAPs regulate caspases. An

RNAi screen of de-ubiquitinating enzymes (DUBs) in Drosophila identified six

pro-apoptotic DUBs, several of which are associated with the ubiquitin-like

modifier NEDD8. They found that IAP family members act as E3-ligases to

“NEDD8ylate” and inactivate endogenous caspases. Domagoj Vucic

(Genentech, San Francisco) presented mechanisms through which

Manchester Cancer Research Centre Conference:Harnessing Apoptosis By Chris Morrow, Kathryn Simpson, Luke Harrison, Tetyana Klymenko, Tom Owens and Fiona Foster

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Eileen White (Rutgers University, New Jersey) described a cell model wherein mTOR inhibition promoted

cell cycle arrest and autophagy but when this autophagy was blocked apoptosis ensued. An anti-cancer

therapeutic strategy might therefore be to stress a cell to make it use autophagy as a survival mechanism

and then inhibit autophagy to drive cell death. Marja Jäättelä (Institute of Cancer Biology, Copenhagen)

introduced a different type of cell death, lysosomal cell death, which can be avoided by cancer cells via the

up-regulation of heat shock protein 70 (HSP70) to enhance the activity of lysosomal acidic spingomyelinase

(ASM) and stabilise lysosome membranes. Inhibition of the interaction of HSP70 and ASM by point

mutation of HSP70, or pharmacological inhibition of ASM prevents lysosome stabilisation to promote cell

death and sensitisation to chemotherapeutics. Considering tumour cells frequently show enhanced ASM

activity, targeting of this enzyme may lead to therapeutic benefit.

Using mice with a knock-in mutant of the PI3-kinase isoform p110a that cannot bind RAS-GTP, Julian

Downward (CR-UK London Research Institute) showed that these mice were less tumour-prone in response

to various RAS-dependent tumourigenic assaults. He also reported a RNAi screen for targets that proved

lethal in a mutant RAS background. Of the hits from this screen, MMP7 showed the most therapeutic

promise, with MMP7 inhibitors combined with topotecan demonstrating selective killing of RAS mutant

cell lines. Christine Watson’s (University of Cambridge) presentation concentrated on studies of the

molecular mechanisms that induced apoptotic cell death during mammary gland involution. STAT3, which

is activated during involution, is one of the key drivers of apoptosis, causing increased levels of the PI3-

kinase subunits p55a and p50a, which may inhibit PI3-kinase activity. STAT3 activation reduced levels of

cathepsin inhibitory protein Spi2a, leading to an increase in cathepsin activity and apoptosis, thus

demonstrating two novel molecular mechanisms for STAT3-mediated apoptosis. Bart Vanhaesebroeck

(Queen Mary University, London) described his elegant mouse models where kinase dead PI3-kinase catalytic

isoforms had been knocked-in to predict effects of specific isoform inhibition by small molecule drugs. Cells

in these mice could proliferate and did not undergo apoptosis, although some isotype knock-ins were

embryonic lethal. However, if multiple isoforms were targeted by genetic and chemical means more


12


Ranson (The Christie NHS Foundation Trust) described findings from the first-in-man trial of AEG35156, a

second generation antisense oligonucleotide targeted to XIAP. In this phase I study AEG35156 was well

tolerated and there was some evidence of anti-tumour activity. Discussion followed regarding the need for

pharmacodynamic biomarkers, as well as the need to determine the tumour drug levels to relate to

therapeutic effect. Caroline Dive (Paterson Institute for Cancer Research) continued the biomarker theme,

speaking on circulating biomarkers of cell death and circulating tumour cells (CTCs). In pilot studies, levels

of circulating cytokeratin 18 and nucleosomal DNA correlated with tumour response and/or toxicity and

had potential for predicting response to standard of care chemotherapy. Interest was generated in the

finding that groups of CTCs (microemboli) have increased ability to avoid anoikis, therefore promoting

metastasis. Gerald Cohen (MRC Toxicology Unit, Leicester) discussed BH-3 mimetic treatment of primary

CLL cells where ABT-737/263 was the most efficacious inducer of apoptosis. Culturing primary CLL cells on

CD154 (CD40L)-expressing fibroblasts to model the lymph node environment induced ABT-737 resistance via

up-regulation of Bcl-xL and Bcl-A1. ABT-263 was ~100 fold less potent in whole blood than ABT-737, an effect

attributed to its greater affinity for serum albumin, demonstrating the importance of modelling the cancer

environment using in vitro models.

Elisabeth de Vries (University Medical Centre, Groningen) provided a comprehensive overview of TRAIL

receptor agonists in Phase I/II clinical trials, with emphasis on the use of recombinant TRAIL, the TRAIL-R1

antibody mapatumumab and five TRAIL-R2 antibodies. These agents were generally well tolerated with no

single agent MTD and no significant toxicity. In phase II studies, single agent activity was reported in some

patients with colorectal cancer, non-small cell lung cancer and non-Hodgkin’s lymphoma.

A vigorous debate at the close of day one over whether apoptosis or autophagy regulatory proteins would

prove the best targets for drug development was

masterminded by Henning Walzcak (Imperial College,

London) championing apoptosis and Kevin Ryan

(Beatson Institute for Cancer Research, Glasgow)

arguing for autophagy research. Henning outlined

therapeutic progress with death receptor agonists, BH3

mimetics, and IAP antagonists. Kevin guided the

audience through the complexities and unanswered

questions surrounding autophagy, highlighting the

paradox that reduced autophagy may lead to

tumourigenesis, while autophagy can also be a survival

mechanism for cancer cells in the harsh tumour micro-

environment. The general consensus at the end of the

debate was that apoptosis targeting agents are

showing clinical promise based on decades of basic

research, while the field of autophagy is still very much

in its infancy and only time will tell whether inhibiting

or activating autophagy mechanisms in tumours may

prove clinically beneficial. The convivial atmosphere and

debate format was enjoyed by all.

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pronounced effects were seen. Mice lacking p110δ were resistant to B16 melanoma tumour take and

metastasis indicative of disabling defects in the tumour stroma.

Rakesh Kumar (George Washington University, Washington) gave an overview of p21 activated kinase (PAK)

that plays several roles associated with tumourigenesis. PAK knock-down inhibited tumour cell invasion

and PAK associated with proteins associated with mitosis. PAK was up-regulated in many breast cancers

where its activity associated with tamoxifen sensitivity, raising the possibility that PAK inhibitors might

sensitise resistant breast cancers to tamoxifen.

Paul Workman (Institute of Cancer Research, Sutton) discussed the translation of small molecule PI3-kinase

inhibition to the clinic. Interestingly for inhibitors of a survival pathway, but consistent with data presented

by Bart Vanhaesebroeck, PI3-kinase inhibition did not induce apoptosis, but rather caused cell cycle arrest.

However, when combined with either temozolomide in glioblastoma cells or TRAIL in colorectal cancer cells,

apoptosis was enhanced. A clinical candidate PI3-kinase inhibitor GDC-0941 had good in vivo activity and

was promising results in a phase I clinical trial, with no dose-limiting toxicities reported. Sylvie Guichard

(AstraZeneca, Macclesfield) completed the signalling pathway session discussing the mTOR inhibitor

AZD8055. This has in vitro and in vivo activity, although whether AZD8055 led to cell death or cell cycle

arrest was cell line dependent. Tumour regression was achieved when AZD8055 was combined with the

MEK inhibitor AZD6244 with biomarkers of apoptosis, again demonstrating that while PI3-kinase pathway

inhibitors may not cause apoptosis per se, there is potential for utilising them in combination with other

drugs to great therapeutic benefit.

The second debate of the conference tackled the virtues and problems of in vitro and tumour xenograft

models versus more complex animal models for anti-cancer drug development. Gerard Evan (University of

Cambridge) reported the benefits of mouse models, in particular the use of genetically engineered mouse

models (GEMMs). The use of these mouse models, in which tumours arise from sporadic in situ oncogene

activation or tumour suppressor inactivation, and evolve through acquisition of secondary lesions, allow

for a more accurate modelling of what occurs in human disease. Donald Ogilvie (Paterson Institute for

Cancer Research) pointed out that a better understanding of the causes or possible treatments of cancer

was our aim and whether the information came from GEMMs or tissue culture models, it could all be

informative. As the representative for the use of tissue culture model systems, Donald highlighted the use

of cancer cell line panels to represent the heterogeneity of the disease even within a given cancer subtype.

At the end of an engaging discussion, GEMMs may have just won the vote, but most agreed that multiple

models were useful and none substituted perfectly for the in situ disease in humans.

The final day started with Jos Jonkers (The Netherlands Cancer Institute, Amsterdam) highlighting the use

of GEMMs. His elegant BRCA1 - p53 null model of breast cancer closely mimics the human disease and

drugs inducing double-strand DNA breaks were shown to be particularly effective. Cisplatin and other

platinum drugs decrease tumour volume, but the tumours continually reoccur without developing

resistance. This would suggest that BRCA1 is required for the generation of resistance to cisplatin. The PARP

inhibitor Olaparib inhibited tumour growth and increased survival but prolonged treatment resulted in

resistance mediated by up-regulation of p-glycoprotein. Currently there are no effective treatments for

pancreatic cancer. David Tuveson (CR-UK Cambridge Research Institute) described his GEMM of pancreatic

cancer, which is indistinguishable from the human disease. This model should allow for the identification

of potential new targets in the development of treatments and novel data on the potentially critical role of

ROS in pancreatic cancer was reported.

The conference was concluded by a plenary seminar from Karen Vousden (Beatson Institute for Cancer

Research, Glasgow) who began with an historical overview of tumour suppressor p53 before focusing on

how the accumulated knowledge of p53 is being translated for clinical benefit. She described recent

research which shed light on role of the mutant forms of p53 in regulating of the motility of malignant cells.

She also discussed how under conditions of mild stress, p53 may contribute to cell survival. By example, she

reported how the p53-inducible protein TIGAR is able to modulate the glycolytic pathway, decrease ROS

levels and consequently inhibit autophagy to promote cell survival.

The conference was a great success, spanning the basic research that continues to unravel the control

mechanisms for cell death to the implementation of apoptotic targeted drugs in the clinic. There was much

excitement about the initial clinical trials with drugs such as ABT-263 and IAP inhibitors, even though it

was clear there was still a considerable amount of work to be undertaken, both at the basic and translational

level, before the full benefit of drugs specifically designed to target cell death pathways are realised.

17

The ‘gold-standard’ method for assessing tumour genotype and phenotype is

an invasive tumour biopsy. However, this often challenging for the patient

and difficult to obtain, particularly serially pre and post drug treatment. This

is particularly problematic in lung cancer patients where tumours may be in

high risk or inaccessible regions of the thorax. In many cases, even where

tissue is obtained, there may be insufficient material for molecular analysis.

Here, CTCs/CTM may represent a ‘virtual biopsy’ readily accessible in real-time

from small volume (2-10ml) blood sample. Serial collection and analysis of

CTC/CTM represents an unprecedented opportunity to study the underlying

mechanisms of the metastatic disease process and compare tumour cells

circulating at disease presentation and at disease relapse. If these circulating

cells can be isolated, purified and characterised (using candidate or global

approaches), this could contribute to the goal of personalised medicine.

Detection and enumeration of CTCs

The number of CTCs present in a patient’s bloodstream varies from less than

ten to several thousand per ml in a background of 10 million leukocytes and

5 billion erythrocytes per ml. Efficient and reproducible purification of CTCs is

therefore the proverbial ‘needle in a haystack’ scenario. Until recently very

few, if any, methods for CTC research demonstrated high specificity and

standardised assay protocols preventing their interpretable use in the clinical

setting and especially in multi-site studies. Enrichment techniques decrease

the degree of normal blood cell contamination and are categorised into i)

positive selection by expression of cell surface markers such as EpCam

(epithelial cell adhesion molecule) and ii) size exclusion methods to capture

cells above a certain size threshold chosen to exclude most blood cells.

Positive selection of CTCs poses obvious limitations if there is heterogeneity

in CTC surface marker expression. Eighty percent of human tumours are

epithelial in origin and as such cells within the tumour mass express markers

such as cytokeratins and EpCam. However, the expression of epithelial

markers is predicted to decrease if CTCs have undergone an epithelial to

mesenchymal transition (EMT) as a prelude to cell invasion as part of

metastatic process.

Lung Cancer Circulating Tumour Cells as Biomarkers

16

Lung Cancer Circulating TumourCells as Biomarkers and aWindow to Metastasis Biology


Cancer metastasis is the predominant cause of treatment failure and cancer

fatality. The metastatic process involves migration and invasion of cancer

cells through tissue, degradation of blood vessel walls and intravasation into

the circulation preceding extravasation and clonal growth in environmentally

primed distant ‘niche’ sites according to the ‘seed and soil’ paradigm. The

identification, enumeration and characterisation of tumour cells in the

circulation has become a pre-eminent focus of the Clinical and Experimental

Pharmacology (CEP) Group led by Caroline Dive, based at the Paterson

Institute for Cancer Research where we a) seek to exploit them as biomarkers

in clinical trials and b) gain insights to circulating tumour cell biology that

might uncover innovative therapeutic approaches and novel targets for drug

discovery. Our recent discovery in lung cancer patients of circulating groups

of tumour cells (micro-emboli) suggestive of an endpoint of collective

migration now drives investigations of anoikis suppression in circulating

tumour micro-emboli and the clinical relevance of epithelial to mesenchymal

transition during lung cancer metastasis.

Characterisation of circulating tumour cells (CTCs) and circulating tumour

micro-emboli (CTMs) is challenging and the biology of tumour cells in the

circulation poorly understood due to paucity of validated methods for their

detection and purification. The Veridex CellSearch™ platform, an

immunomagnetic based method for CTC enumeration, has galvanised this

field of research with numerous studies demonstrating utility of CTCs as

prognostic biomarkers. It is widely used clinically, is reproducible and able to

detect CTCs across a range of tumour types inculding breast, colorectal,

prostate, ovarian, bladder and lung. The US Food and Drug Administration

(FDA) approved this technology to monitor progression-free and overall

survival in breast, colorectal and prostate cancers. This and other advances in

technology are now heralding a step-change in our ability to qualify CTC/CTM

biomarkers that predict and/or monitor response to treatment, identify

potential therapeutic targets, and unveil and understand heterogeneity in

tumour cells that survive the circulation to promote metastasis.

By Tim Ward, Jian-Mei Hou, Matthew Krebs, Fiona Blackhall and Caroline Dive

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Circulating tumour cells and microemboli: collective migration, anoikis and epithelial

mesenchymal transition.

Metastatic spread is postulated to occur via Paget’s ‘seed and soil’ paradigm and via epithelial to

mesenchymal transition (EMT) where down-regulation of epithelial and up-regulation of mesenchymal

cellular characteristics facilitate invasive behaviour of single tumour cells. More recently, the process of

collective migration of cancer cells through tissue has been proposed where clusters or strands of cells

invade through tissues cooperatively,- although the epithelial versus mesenchymal phenotype of these cell

groups has not been reported. In addition, preclinical models suggest a co-operation between epithelial and

mesenchymal cells during the metastatic process that somewhat challenges the strict definition of the

EMT paradigm. In a recent and pilot study of NSCLC and SCLC patients, we identified lung cancer patients

in whom cancer cells circulate as CTM and as single circulating tumour cells (CTCs). CTM were composed

of clusters and strands consistent with the morphologies described for collective migration. Considerable

intra- and inter-patient heterogeneity was observed in EMT markers (vimentin, E-Cadherin, N-Cadherin,

cytokeratin) consistent with incomplete EMT or with co-operation between epithelial and mesenchymal

tumour cells. CTCs, but not cells within CTM, exhibited apoptotic nuclei consistent with the hypothesis

that tumour cells circulating within CTM are more likely to suppress anoikis (the engagement of apoptosis

upon loss of cell-cell contacts and extracellular matrix). Further studies are underway to explore the clinical

significance of these early findings. Venous thromboembolism (VTE) is a well known complication of

metastatic cancer and the presence of tumour cells in the circulation has been associated with increased

risk of VTE in metastatic breast cancer patients. Prospective studies are now underway in the CEP Group to

understand the clinical significance of lung cancer CTM in addition to the molecular mechanisms driving

their behaviour that may provide new insights for therapeutic control.

18


The CEP Group employs the Veridex CellSearch™ approach to enumerate CTCs in clincal trial settings and

the CellScreen ISET platform, a filtration based approach, in research mode to characterise CTCs and CTMs.

The Veridex CellSearch™ platform is a semi-automated system that enriches for CTCs using a ferrofluid

medium of magnetic particles in an EpCAM polymeric coated layer, the EpCAM acting as the capture

antibody. Cells remain intact allowing some morphology assessment and enumeration and CD45

expression is used to identify and exclude leukocytes. CTCs are confirmed by their expression of cytokeratins

(8,18,19) as tumour specific markers. DAPI staining allows assessment of apoptosis via condensed and/or

fragmented nuclear morphology. Enrichment is in the order of 104 to 2x105 fold and sensitivity is one cell

per 0.5ml blood. The fixed and stained cells are washed and dispensed into a cartridge which is inserted into

a ‘MagNest’ device. This attracts cells to a position within a magnetic field and an inbuilt flouresecent

microscope scans this position to produce flourescent images in a gallery format for a manual decision on

what is or is not a CTC. There is an additional spare channel in the standard Veridex CTC kit that allows

examination of a fourth molecule of interest, for example a drug target or pharmacodynamic biomarker. As

examples, the CEP Group is currently examining CD56 (a neuroendocrine marker in small cell lung

carcinoma (SCLC) cells), and Bcl-2 or Mcl-1 (potential biomarkers of senstivity or resistance to the BH-3

mimetic drug ABT 263 respectively). Further analysis is now possible on CTCs recovered from the cartridge

after initial analysis using multi-colour fluorescence in situ hybridisation (FISH). The CEP Group is using this

approach to look for abnormalities in bcl-2 and c-myc gene copy number in SCLC CTCs.

Using the CellScreen ISET platform, blood is filtered through membranes with 8µm pores. Leukocytes are

smaller than CTCs and the vast majority pass through the filter. A blood sample is collected in an EDTA

tube, diluted with buffer to fix the cells and then filtered under vacuum. The system has been shown to

isolate one cell per 1ml blood and successful gene amplification has been a achieved with as few as five

cells per filter. Cytological staining and immunohistochemistry (IHC) can then be performed and the cells

enumerated and characterised. It is also possible, with careful method development, to use the enriched

CTCs for nucleic acid extraction and subsequent genomic analysis and these are ongoing and future

developmental projects in the CEP Group.

Circulating tumour cells in lung cancer patients – a primary focus of the lung cancer disease focus

group in CEP

Lung cancer is a leading cause of cancer related deaths worldwide. Non-small cell lung carcinoma (NSCLC)

lacks validated biomarkers to predict or provide early reporting of treatment response. The CEP Group

investigated whether CTCs are detectable in patients with SCLC and NSCLC and whether they could provide

prognostic information and/or early indication of response to conventional therapy. A high number and

wide range of CTCs were detected using Veridex CellSearch™ in SCLC and the CTC number was both

prognostic and pharmacodynamic. Blood samples were assessed using the Veridex CellSearch™ system for

CTC analysis from 100 patients with previously untreated stage III/IV NSCLC, before and after administration

of one cycle of standard chemotherapy. The CTC number was higher in patients with more advanced stage

IV compared to stage III patients. In multivariate analysis, CTC number was the strongest predictor of overall

survival (OS). This study clearly demonstrated that CTCs are detectable in patients with stage IV NSCLC

where their number is a novel independent prognostic factor.

Lung Cancer Circulating Tumour Cells as Biomarkers

Figure 1. Characterisation ofCTCs. CTCs isolated by eitherthe CellSearch Technology orthe ISET platform were furthermolecularly characterised.Figure A shows a single CTCisolated from SCLC patientwith high nuclear/cytoplasmicratio, bigger cell size (the poreon the membrane is 8 µm indiameter) and irregularnuclear shape. Figure B showsSCLC CTM (black arrow)isolated by ISET and negativelystained for CD45 whereasleukocytes were positivelystained (white arrow). FigureC shows that CTCs detectedfrom SCLC patients can befurther characterisedregarding tumour specificmarker (Ci, CD56), potentialdrug target (Cii, Bcl-2), or drugresistant marker (Ciii, Mcl-1) bythe 4th channel of the VeridexCellSearch™ platform.Similarly SCLC associatedmarkers, NSE and TTF-1 werestained to further identify ISETisolated CTM and CTC (FigureD). Figure E illustrated thatBCL2 FISH analysis can detectBCL2 genetic abnormalitiessuch as amplification andtriploidy from SCLC CTCs.

21

division), transcription (the conversion of DNA into RNA in advance of protein

synthesis by translation) and mitosis (the segregation of chromosomes within

the cell nucleus into two identical daughter nuclei). Poly(ADP-ribose) (PAR) is

a highly negatively-charged polymer that is formed from repeating ADP-ribose

units linked via glycosidic ribose-ribose bonds, and is synthesised by the

poly(ADP-ribose) polymerase (PARP) family of enzymes using a vital cellular

cofactor NAD as a substrate (Figure 1). The recent development of potent PARP

inhibitors has provided powerful tools to study the pathways regulated by

poly(ADP-ribose), as well as providing a very promising class of drugs for

cancer treatment. Specifically, selective inhibition of the single-strand break

repair pathway using permeable PARP inhibitors has been proven to be highly

effective against breast and ovarian cancers. More recent data suggest that

the same inhibitors might be effective against several other cancer types as

well and PARP inhibitors have already entered phase III of clinical trials.

DNA Damage Response

20

DNA Damage Response


DNA is constantly exposed to damage and living organisms have evolved a

variety of DNA repair mechanisms to maintain genome stability. Inadequate

or abnormal DNA repair can cause diseases that in humans are associated

with cancer, neurodegeneration, immunodeficiency or developmental

abnormalities. Therefore, furthering our understanding of the molecular

pathways employed in the mammalian response to DNA damage potentially

provides a basis for the development of new therapies in the treatment of

human disease.

Many cancer therapy procedures, such as radiotherapy and some types of

chemotherapy, work by overwhelming the capacity of the cell to repair DNA

damage, resulting in cell death. Most rapidly dividing cells - cancer cells - are

preferentially affected by such treatments, providing the opportunity to use

DNA damaging agents to selectively kill cancer cells. In addition, the

development of cancer is underpinned by genomic instability and the

accumulation of multiple DNA mutations resulting in the loss of cellular

growth control. In order to accelerate the accumulation of these genetic

changes, cancers often sacrifice specific DNA repair pathways. This can make

cancer cells additionally susceptible to DNA damaging agents and/or to

inhibitors that block alternative repair pathways that allow cancers to thrive

without the full DNA repair repertoire. For these reasons, studying the protein

components involved in repair of damaged DNA has been proven a valuable

strategy in searching for novel approaches and targets in cancer therapy.

There are many pathways and signalling strategies for counteracting the

deleterious effect of DNA damage in humans. In our laboratory we are

particularly interested in studying the DNA repair pathways and protein

functions that are regulated by poly(ADP-ribosyl)ation. Poly(ADP-ribosyl)ation

is a post-translational protein modification employed by several important

nuclear processes including DNA repair, regulation of chromatin structure

(the combination of DNA, RNA, and protein that makes up chromosomes),

cell cycle checkpoint (control mechanisms that ensure the fidelity of cell

By Ivan Ahel

Figure 1. Regulation of DNA damage response by poly(ADP-ribosyl)ation.

22


The principal poly(ADP-ribose) polymerase involved in DNA repair is called PARP1. PARP1 is a DNA damage

sensor protein that specifically recognises breaks in DNA structure, including both single-strand and double-

strand DNA breaks. Upon binding to DNA, PARP1 catalytic activity is activated to produce vast amounts of

poly(ADP-ribose), which makes the DNA damage response a major source of cellular poly(ADP-ribose)

synthesis. The poly(ADP-ribose) produced by PARP1 is covalently attached to many target proteins including

PARP1 itself, histones and tumour suppressor protein p53, which allows the regulation of several different

aspects of the cellular response to DNA damage (Figure 1). Firstly, the local increase of poly(ADP-ribose)

arising at the sites of damaged DNA serves as a platform for the protein complexes involved in trimming

and sealing DNA breaks. In other words, many of the protein factors associated in such DNA repair factories

possess specialised protein modules to specifically bind to poly(ADP-ribose), which allows their timely

recruitment. Another consequence of signalling by PARP1 is the relaxation of chromatin structure via the

displacement of histones at the damaged area. This is particularly important as the chromatinised DNA is

tightly packaged due to it being wrapped around the histones, which in turn makes it refractory to efficient

repair. Finally, excessive poly(ADP-ribosyl)ation as a consequence of DNA damage beyond the cellular repair

capacity is a powerful signal for programmed cell death by apoptosis. Apoptotic signalling is critical for

complex organisms, as it prevents mutations and the development of cancer. Taken altogether, poly(ADP-

ribosyl)ation regulates several key events in DNA damage response and thus is essential for genomic

stability. However, many of the protein components involved and their mechanisms of regulation remain

unknown.

In our laboratory, we are routinely screening for novel proteins that have the ability to respond to DNA

damage in a manner that can be inhibited by treatment with PARP inhibitors. For this, we are using real-

23

time in vivo imaging by confocal microscopy, in combination

with a state-of-the-art laser system, that allows the infliction of

spatially controlled DNA damage to the cell nucleus and

subsequent analysis of the mobilisation of fluorescently

labelled proteins (Figure 2). Utilising this approach we have

recently discovered a novel structural element associated with

several DNA damage response factors which we have named a

poly(ADP-ribose)-binding zinc finger (PBZ) (Figure 3). PBZ is

distinctive of canonical DNA-binding zinc fingers and it is used

by proteins to recognise and bind to poly(ADP-ribose) with a

high affinity. One of the human proteins containing a PBZ

motif is a protein called Checkpoint protein with FHA and RING

domains (CHFR). CHFR is an ubiquitin ligase frequently

inactivated in human epithelial tumours, which acts as a key

modulator of the early mitotic checkpoint (this checkpoint

transiently delays mitosis in response to a variety of stresses). The elucidation of the function of the PBZ

motif gave us a vital clue to discover that the CHFR-dependent checkpoint is regulated by PARPs and that

the PBZ motif in the CHFR protein is critical for checkpoint activation. Another PBZ-regulated protein we are

studying is a protein called Aprataxin-PNK-like factor (APLF). APLF is constitutively associated with the DNA

repair ligases (enzymes responsible for sealing the breaks in DNA) and its down-regulation leads to an

apparent cellular sensitivity to various DNA damaging agents. However, the exact function of APLF in DNA

repair is presently unclear.

Another class of proteins that our research focusses on is macro-domain proteins. The macro-domain is

another module with the capacity to bind poly(ADP-ribose), and the aberrant regulation of several human

macro-domain containing proteins has already been linked to the development of cancer. One of these

proteins is a putative ATP-dependent helicase ALC1 (Amplified in Liver Cancer; also know as CHD1L), which

is frequently over-expressed in human hepatocellular carcinoma (HCC). The role of ALC1 in tumorigenesis

appears to be direct, as uncontrolled expression of ALC1 in mice leads to cancer development. In our recent

work, we provided the molecular evidence that ALC1 acts as a PARP1-regulated chromatin remodelling

enzyme in response to DNA damage and that its histone-repositioning activity is required for efficient DNA

repair. Strikingly, over-expression of ALC1 leads to deregulation of DNA damage signalling pathways and

results in a specific sensitivity to certain types of DNA-damaging drugs. These findings emphasise the

importance of chromatin reorganisation in DNA repair as a significant element in genome stability and

suggest a potential basis for developing a targeted therapy for ALC1-overexpressing tumours.

In conclusion, our research aims to better understand the basis of the response to DNA damage through the

identification of novel molecules that may play a role in this response. Exploring the regulation and function

of these molecules will allow us to establish whether and how they may impact cancer development. We

hope that our fundamental research approach will facilitate translational research via the identification of

potential molecular targets for the development of rational therapeutic interventions.

DNA Damage Response

Figure 2. Recruitment of fluorescently labelled ALC1 protein to the laser-induced DNA break sites in the cellnucleus. The recruitment is blocked by treatment of cells with the specific PARP inhibitor (lower panel).

Figure 3. The structure of the poly(ADP-ribose)-binding zinc finger

25

Projects

In order to make a valuable contribution to cancer drug discovery, we

deliberately work in project areas that are overlooked or currently considered

too risky by the major pharmaceutical companies. This will include for

example, segments of cancer with smaller patient numbers or novel biological

concepts in which the MCRC has world-leading expertise. During late 2009

we presented our cancer drug discovery target “roadshow” to many groups

of scientists and clinicians in the Paterson Institute, The Christie Hospital and

The University of Manchester. These presentations have been followed up

with more detailed discussions and this has allowed us to identify and

prioritise our first drug discovery projects. These projects are focused around

modulation of specific target molecules, in cells or tissues that we believe to

be involved in driving malignant tumour growth and progression. Target

review will be an ongoing activity so that we can keep abreast of new

developments in cancer science and fuel the drug discovery “pipeline” with

the best opportunities.

Once a target is chosen, the aim of drug discovery is to identify and optimise

chemical agents that selectively interfere with the target activity in order to

kill or restrain the tumour cells, without unacceptable effects on normal

tissues. This is an iterative process involving the identification of initial

chemical “hits”, the exploration of their drug potential to create “leads” and

then the optimisation of these leads to create a clinical candidate for testing

in cancer patients. There are several approaches to hit identification which

include screening of existing compound collections and generating and

exploiting detailed structural information on the target. Selection of the final

clinical candidate chemical compound includes optimisation of not just target

modulation but also selectivity, to avoid unwanted side effects, and delivery

to the required site(s) of action in the body. During 2010 we initiated four

MCRC hit identification projects and also engaged in successful high

throughput screening and lead identification collaborative projects with the

CRT Development Laboratory in London

Drug Discovery in the Manchester Cancer Research Centre

24

Drug Discovery in theManchester Cancer Research Centre


In 2009, a £9 million Cancer Research UK five-year programme grant was

awarded to the Paterson Institute for Cancer Research to build a cancer Drug

Discovery Unit in the MCRC. This new initiative arose from a strategy review

in which Cancer Research UK decided to increase significantly their long term

investment in small molecule drug discovery and to align this additional

resource with the core-funded cancer research institutes in Glasgow (Beatson

Institute for Cancer Research) and Manchester (Paterson Institute). The

purpose of co-locating these activities is to maximise the opportunity for

translating the ground-breaking cancer research from these centres of

excellence into novel therapeutic opportunities.

The ultimate aim of the MCRC Drug Discovery Unit is to identify novel drug

therapies to satisfy some of the many unmet clinical needs of cancer patients.

However, drug discovery and clinical development are long and complex

processes and in order to achieve this goal we will need to capitalise on the

outstanding opportunities afforded by the MCRC environment.

During 2009-10, the foundations of the MCRC Drug Discovery Unit, including

strategy, facilities and key recruitment, were laid and the first drug discovery

projects were started.

At the outset, strategic considerations included: 1) What kind of drug

discovery projects should we prosecute in order to make a valuable, unique

contribution to the global war against cancer?, 2) What kind of drug discovery

skills should we focus our limited resources on?, 3) What kind of laboratory

facilities do we need to support these activities? and 4) What kinds of

partnerships, within and beyond the MCRC, do we need to complement our

in-house capabilities in order to achieve our long term objectives?

By Donald Ogilvie and Allan Jordan

27

Partnerships

The first key partner is of course Cancer Research UK who are providing the crucial funding - £9 million for

the first five years. But Cancer Research UK is more than just a source of funding for this new venture. As

well as individual programme grants, Cancer Research UK already supports major Drug Discovery Units in

London, Sutton and Newcastle providing a broad portfolio of projects. The new Units in Manchester and

Glasgow are seeking to complement one another in extending this portfolio into new areas of breaking

cancer science and drug discovery technology. The leaders of these Drug Discovery Units meet regularly to

share expertise, coordinate their activities and identify areas of cooperation and collaboration in order to

maximise the effectiveness of Cancer Research UK drug discovery. As one example of this cooperation, we

are accessing the compound collection and screening technology in the London Unit (CRT-DL) to support our

hit identification projects. Another important part of the Cancer Research UK “family” is Cancer Research

Technology (CRT) which provides us with intellectual property and business development support. These

activities are particularly important for facilitating collaborations, the protection of drug discovery

inventions and, in the longer term, for identification of partners to take our candidate drugs into clinical

trials.

MCRC

Our location in the Paterson Institute, at the heart of the MCRC, is ideal for accessing clinical insight, basic

research expertise and world-leading clinical development technologies and experience. A key component

of the MCRC is the breadth of clinical expertise at The Christie NHS Foundation Trust. This provides direct

insight into the areas of unmet clinical need and the hypotheses to address them but also brings a tangible

connection with our ultimate customer, the cancer patient. At the other end of the MCRC spectrum are the

basic scientists in the Paterson Institute and more broadly in The University of Manchester who provide

insights into the mechanisms of cancer and how to measure these in preclinical models. In the middle are

the translational scientists and clinicians, particularly in the Clinical and Experimental Pharmacology Group

at the Paterson Institute, who provide the roadmap for initial clinical development, particularly in the

validation of novel biomarkers. We are also exploring opportunities to access other key technologies (for

example biophysical chemistry, biochemistry and protein structural analysis) through experts in The

University of Manchester.

Since drug discovery and development takes such a long time (10+ years) and many projects do not progress

to clinical trials we need to need to be able to demonstrate that we are making progress in the shorter term.

In the first five years, this will be primarily through the generation of a unique (within Cancer Research UK)

portfolio of attractive drug discovery projects.

During the first two years of our operation we have laid the foundations of the new MCRC Drug Discovery

Unit and have initiated and advanced our own portfolio of novel drug discovery projects. During the next

period we aim to enhance and progress this portfolio towards the goal of clinical testing of innovative cancer

medicines.

Drug Discovery in the Manchester Cancer Research Centre

26


Skills

Our core expertise is in the areas of cancer drug target selection and drug discovery biology and chemistry

and this focus has been reflected in our recruitment strategy. During the summer of 2009, Allan Jordan

joined the team as Head of Chemistry. Towards the end of the same year, a major campaign was successfully

carried out to recruit a further seven scientists (medicinal/synthetic chemists and biologists) with

pharmaceutical industry experience to kick start the laboratory work from early 2010. All other kinds of

expertise, including clinical insight, biomarkers and other bioscience technologies are being accessed

through collaborations, partnerships and outsourcing as appropriate (see below). Towards the end of 2010

a second wave of recruitment was initiated to expand the team and broaden our skills base to include

computational chemistry and drug metabolism expertise.

Facilities

The major activity during 2009 was the design and construction of a modern drug discovery laboratory.

With the help of external advisers, the Paterson Institute estates team and an excellent building contractor,

the new laboratory was designed and completed on time and handed over in December 2009. An unusual

feature of the design is the co-location of biology and chemistry facilities. This was a deliberate decision to

ensure close interdisciplinary interactions in the design, synthesis and testing of new compounds. Parallel

procurement of key capital equipment ensured that the new laboratory was able to open for work in January

2010. A major project during 2010 has been the construction and optimisation of the informatics

infrastructure including Virtual Screening, Electronic Notebooks and the Dotmatics chemoinformatics

platform. The latter enables tracking of the complete data workflow from novel compound design and

synthesis to biological testing.

29

(discriminatory power). Currently the lifetime risk for a woman in the UK of

developing breast cancer is between 9 and 11%; if she has no family history of

breast cancer the risk is lower at around 8%. Three high risk genes have been

identified to date which predispose to breast cancer in women - TP53, BRCA1

and BRCA2 with the following risks:

• TP53 – 1990, lifetime risk 90%

• BRCA1 – 1994, lifetime risk 60-85%

• BRCA2 – 1995, lifetime risk 50-85%

There are two main types of risk assessment; 1) the chances of developing

breast cancer over a given time-span including lifetime and 2) the chances of

there being a mutation in a known high-risk gene such as BRCA1 or BRCA2.

Whilst some risk assessment models are aimed primarily at solving one of

the questions many also have an output for the other. For instance a

computer algorithm BRCAPRO is primarily aimed at assessing the mutation

probability but can have an output to assess breast cancer risk over time. The

Cuzick-Tyrer model was developed to assess breast cancer risk over time but

does have a read out for BRCA1/2 probability for the individual. In order to

assess breast cancer risks over time as accurately as possible all known risk

factors for breast cancer need to be assessed.

Risk factors

Family history of breast cancer in relatives

• Age at onset of breast cancer

• Bilateral disease

• Degree of relationship (first – for example mother, sister

or greater – for example aunt, cousin)

• Multiple cases in the family (particularly on one side,

for example the maternal lineage)

Genetic Medicine and the Genesis Prevention Centre

28



The main focus of cancer research in Genetic Medicine has been on genetic

predisposition to breast cancer and neurofibromatosis. Whilst the hunt

continues for a fourth high risk breast cancer gene, genome-wide association

studies, to which we have been a major contributor, continue to find common

genetic variants that enhance risk of breast, colorectal and other cancers. We

have been using these variants to assess variation of risk amongst different

populations. Through an NIHR programme grant we are collecting DNA

samples in the high risk clinic as well as from a group of 60,000 women being

recruited from the National Breast Screening Programme.

Breast Cancer

The authors have published over 200 papers on inherited predisposition to

and aetiology of breast cancer in the last 15 years.

Risk prediction

Breast cancer is diagnosed in 44,000 women and causes 12,000 deaths per

year in the UK. Although breast cancer deaths have decreased in many

Western countries, the incidence of the disease is continuing to rise. In

particular in countries with historically low incidence, breast cancer rates are

rising rapidly making it the world’s most prevalent cancer. The increase in

incidence is almost certainly related to dietary and reproductive patterns

associated with Western lifestyles. Indeed there is evidence from genetic

studies in the US, Iceland and UK of a 3-fold increased incidence in the general

population and also in those at the highest level of risk with BRCA1/2

mutations in the past 80 years. Understandably there is increasing interest

in disease prevention to spare women the trauma of diagnosis and

increasingly aggressive treatment. There is a need, not only to predict which

women will develop the disease, but also to apply drug and lifestyle measures

in order to prevent the disease. Current risk prediction models based on

combinations of risk factors have good overall predictive power, but are still

weak at predicting which particular women will develop the disease

By D Gareth Evans and Anthony Howell

31


30


• Other related early onset tumours (for example ovary, sarcoma)

• Number of unaffected individuals (large families with many unaffected relatives will be less likely

to harbour a high-risk gene mutation)

Hormonal and reproductive risk factors

Hormonal and reproductive factors have long been recognised to be important in the development of breast

cancer. Prolonged exposure to endogenous oestrogens is an adverse risk factor for breast cancer. Early

menarche and late menopause increase breast cancer risk as they prolong exposure to oestrogen and

progesterone.

Long-term combined HRT treatment (>5 years) after the menopause is associated with a significant increase

in risk. However, shorter treatments may still be associated with risk to those with a family history. The risk

from oestrogen-only HRT appears much less and may be risk-neutral. A meta-analysis also suggested that

both during current use of the Combined Oral Contraceptive and 10 years post use there may be a 24%

increase in risk of breast cancer.

The age at first pregnancy influences the relative risk of breast cancer as pregnancy transforms breast

parenchymal cells into a more stable state, potentially resulting in less proliferation in the second half of the

menstrual cycle. As a result, early first pregnancy offers some protection, whilst women having their first

child over the age of 30 have double the risk of women delivering their first child under the age of 20 years

and these are likely to be similar in those at highest risk from a BRCA1/2 mutation.

Other risk factors

A number of other risk factors for breast cancer are being further validated. Obesity, diet and exercise are

probably interlinked. Mammographic density is perhaps the single largest risk factor that is assessable but

may have a substantial heritable component. Other risk factors such as alcohol intake have a fairly small

effect and protective factors such as breast-feeding are also of small effect unless a number of years of

total feeding have taken place. None of these factors are currently incorporated into available risk

assessment models

Risk factors included in current models

Current risk prediction models are based on combinations of risk factors and have good overall predictive

power, but are still weak at predicting which particular women will develop the disease. New risk prediction

methods are likely to come from examination of a range of high-risk genes as well as single nucleotide

polymorphisms (SNPs) in several genes associated with lower risks. These data would be married in a

prediction programme with other known risk factors to provide a far more accurate individual prediction.

-

Predicting risk of breast cancer at screening (PRoCAS)

PRoCAS is part of a £1.59 million programme grant from NIHR which started in 2009. The overarching aims

of the programme are to improve methods for prediction of risk of breast cancer in order to develop risk

adapted mammographic screening and targeted preventive strategies.

We aim to improve the precision of prediction of breast cancer risk in women in the NHS Breast Screening

Programme (BSP) by combining risk factors into existing programmes and investigating new genetic

modifiers of risk. We will also assess the viability of reducing or increasing screening intervals in the BSP

based on risk algorithms.

0

1

2

3

4

5

6

7

Breast density

Weight gain

Plasma E

2

Late first birth

Bone density

Late menopause

Waist hip ratio

HRT E+PM

enarche <12yrsExercise

Rel

ativ

e R

isk

Potential risk factors

Lifetimerisk25%

Lifetimerisk4%

Dense breast Non dense breast

Mammographic Density

33


32


Research plan

New risk prediction methods will be developed from examination of a range of SNPs for high-risk as well

as those associated with lower risks. This will be married in a prediction program with other known risk

factors to provide greatly improved discriminatory power and accuracy of individual risk prediction. We will

include information on mammographic density which is not yet part of a risk prediction model despite

being a greater risk factor than most factors currently included. Risk assessment of women in the BSP will

determine appropriate screening intervals, or if they are at sufficient risk for BSP screening with huge

potential savings to the NHS from more focused use of resources. Importantly for a group of 10,000 women

we are also going to use DNA extracted from a saliva sample to help predict risk. We anticipate the

development of highly predictive algorithms for risk prediction within five years and to have developed

validated prevention programmes appropriately targeted at risk.

Environment

The programme is based in a new £14m breast cancer treatment and prevention centre, The Nightingale

Centre & Genesis Prevention Centre at the University Hospital of South Manchester NHS Foundation Trust,

and builds on development work from ourselves. This is the largest family history clinic and screening

population in the UK. Dissemination to the network will be through our links to Greater Manchester Clinical

Research Network and BSP.

FHrisk protocol

By studying 3,170 women who were unaffected at the time of assessment we previously showed that the

Tyrer-Cuzick model was most accurate at predicting which women would develop the 84 breast cancers

that occurred. This remains the only such validation in the family history clinic (FHC) setting worldwide. We

have now expanded our FHC set to over 8,900 and are already aware of 315 breast cancers that have

occurred. By matching details against the North West Cancer Intelligence Service on 1 December 2009 we

will obtain an accurate assessment of cancer status and predict that a total of 350 breast cancers will have

occurred by that date. Whilst models have good overall predictive power, they are still less good at predicting

which particular women will develop the disease. We will therefore revalidate the original models alongside

a new model - BOADICEA - in this expanded FHC set. We already have risk information on extended family

history, age at menarche, hormonal treatments, age at menopause, and previous breast disease to enter into

the various risk programmes. We will then use information on breast density from mammography and

body mass index (BMI) to improve the predictive value of the best model(s). Data from genetic testing will

also be included with BRCA1/2 information already known on close to 3,200 women. We will obtain DNA

from blood samples on three controls for every breast cancer patient and validate the new genetic variants

being identified. The expected improvements in risk prediction for women will enable more appropriate

targeting of screening and preventive measures.

Neurofibromatosis type 2 (NF2)

NF2 affects the skin and nervous system (including the brain) and is characterised by skin lesions and benign

tumours – neurofibromas – which most commonly occur on the acoustic nerves leading to symptoms

including deafness. Between 1990-92 we carried out the original large clinical and genetic study of NF2. This

research was the definitive description of the epidemiology and genetics of the condition. We were the

first to describe the high level of mosaicism in NF2 and the original evidence for a genotype-phenotype

correlation. We have also published the definitive paper on mortality in NF2, which showed worse survival

in non-specialist units. We have now secured a national bid from the National Commissioning Group for

the management of type 2 neurofibromatosis for £7.1 million annually, which starts in April 2010. We have

published over 80 articles on NF2 that show the importance of multidisciplinary management and these

have shown a survival advantage for patients so managed. This led to a consensus conference at Old Trafford

in 2004, which we organised and chaired, and lead to the publication of national consensus guidelines.

Invitation letter sent

Consent taken & questionnaire completed

Mammogram 1 performed

Initial risk calculation (Tyrer-Cuzick)

Mammogram 2 performed

Breast density results, questionnaire results & DNA results(if applicable) combined to give re-adjusted risk score

OPTIONAL - DNA sample collected (10,000/60,000)

High risk & selection of low risk women informed of risk(if opted to receive risk information)

Flowchart

35

complexity at the DNA level than other organisms. The resolution of this

paradox has come from studies of how the DNA is expressed, and how the

products of gene expression (RNA and proteins) are produced, regulated, and

processed.

The early view of a gene was of a single contiguous piece of DNA that was

transcribed into a messenger RNA (mRNA) intermediate, which in turn was

translated into a functional protein. This simple view has, over the past 40

years, transpired to be immensely complex. A typical gene is now known to

be split into units called exons - sections of the gene which ultimately are

Cancer Genomics in the Era of High Throughput Sequencing

34


Cancer Genomics in the Era ofHigh Throughput Sequencing

Manchester Cancer Research Centre Annual Research Report

When the first working draft sequence of the human genome was published

in 2000, it was the culmination of a massive international effort that some

have likened to that of putting a man on the moon. The Human Genome

Project took thirteen years to complete at a cost of more than of $3billion.

Now, under ten years later, advances in technology are making it possible to

assemble similar amounts of raw sequence in a few weeks using a single

machine. The relatively low cost - currently under $10,000 per genome -

makes it feasible to use the technology for many purposes, ranging from

sequencing the complete genomes of individuals, through characterising

panels of human tumours to detect common mutations, to analysing all the

regions of the genome that are actively transcribed. A variety of competing

technologies exist, each with their own strengths and weaknesses. Within

the Molecular Biology Core Facility (MBCF) at the Paterson Institute for Cancer

Research, we have recently taken delivery of an AB SOLiD™ machine that is

capable of generating over a quarter of a billion residues of high quality

sequence a week. This report will highlight some of the current applications

of this new high throughput sequencing technology (HTS), as well as

describing other platforms that have been established within the Paterson

Institute and which have been widely used within the MCRC.

Each human cell contains 23 pairs of chromosomes comprising 3 billion base

pairs of DNA. Within this DNA are all the determinants for human

development, ageing and, potentially, disease. Until the entire sequence of

the human genome was produced, we had no idea of the exact number of

human genes, and in many cases no clue about their function. Surprisingly,

man was found to have similar numbers of genes to many apparently simpler

organisms such as flies and worms; paradoxically, a very complex and

intelligent organism appeared to have no significantly greater genetic

By Crispin Miller, Jenny Varley and Stuart Pepper

Exon 1 Exon2

Exon 3 Exon 4

Intron 1 Intron 2 Intron 3

Transcription

Alternative Splicing

Translation

Post-translational modification

Promoter

Figure 1. Cartoon showing a simplified structure of a typical gene. At the top of the figure is a stylisedrepresentation of a gene showing the position of a promoter where transcription into the precursor RNA starts,four exons and three introns. After transcription into the pre-RNA introns may be alternatively spliced; in thiscartoon two alternatives are shown. Upon translation two different but related proteins are produced,differing due to the presence or absence of exon 2 material in the transcripts. Finally the proteins may bemodified post-translationally as indicated at the bottom of the figure. In this highly simplified cartoon threedifferent protein products are produced from a single gene.

37

when considering rare types of tumours. The use of FFPE

material, some of which may be many years old, also

allows us to study gene expression in tumours for which

there is information on patient clinical outcome, after

many years’ follow up (Figure 2).

One major use of genomic technologies is the analysis of

DNA from multiple samples, supporting searches for the

polymorphisms and mutations that underpin diseases

such as cancer. There is considerable variability in the

DNA sequence between different individuals

(polymorphisms), and this variability in key regions of the

genome may predispose to diseases such as cancer, as

well as others such as heart disease, diabetes,

neurological and ageing disorders. Not only may normal

variations in our DNA predispose to cancer (for example

by altering the way our cells deal with carcinogenic

agents such as those found in tobacco smoke), but mutations in specific genes within our tissues may be

responsible for the development or progression of a cancer. In addition, many tumours are characterised

by the amplification of some genes and the loss of others, leading to their over- or under-expression. There

is therefore a strong impetus to determine as much as we can about the normal DNA from cancer-prone

individuals as well as investigating, in an unbiased fashion, what genetic changes are occurring in the actual

cancer cells. We can currently carry out this analysis using one of two platforms. Firstly, we can interrogate

Affymetrix SNP GeneChips® in which nearly one million single nucleotide polymorphisms (SNPs) and the

same number of copy number variants (CNVs) are arrayed. Although GeneChips are a very powerful tool for

DNA and expression analysis, the investigations are limited to those regions of the genome represented by

probes on these commercially available arrays. The second platform that can be used to analyse SNPs and

CNVs is the HTS platform which provides unbiased information by analysing the entire genome of individual

tissue or tumour samples.

However, it would be wrong to focus solely on DNA-level analyses when considering the impact of HTS

technology. A consequence of the sheer amount of data it can generate is that it can be used in a wide

variety of assays that require massively parallel DNA or RNA identification. For example, in RNA-Seq studies,

messenger RNA is extracted from a sample, sequenced, and aligned to the genome, before counting the

number of sequences that match to the location of each gene. The result is a global picture of gene

expression.


36


translated and transcribed into the mature protein - separated by introns, which are removed to produce the

mature mRNA transcript, and are therefore not translated. Furthermore, after the gene is translated into a

precursor RNA molecule, there may be multiple exons that can be joined (spliced) in a variety of different

ways, a process known as alternative splicing (Figure 1). Alternative splicing is therefore the mechanism by

which cells can remove different sections of an RNA molecule in a highly coordinated and tightly-regulated

fashion, allowing the expression of a series of distinct, but related, proteins from a single gene. Originally

discovered in the 1970s and initially viewed as a rare event, alternative splicing is now known to affect the

majority of human genes. It allows cells to substantially increase their repertoire of available proteins, and

helps to explain why man has a similar number of genes in comparison to other, apparently simpler,

organisms. Not surprisingly given the genetic basis of cancer, patterns of splicing are markedly different

between tumours and normal cells, and can be indicative of, for example, patient prognosis.

We have recently been using Affymetrix GeneChip® Exon Arrays to identify patterns of alternative splicing.

These arrays feature probesets that target every known and predicted human exon in the genome. One

study that has been carried out as a collaboration between Doctor Crispin Miller (Applied Computational

Biology and Bioinformatics (ACBB) Group at the Paterson Institute) and Professors Catharine West

(Translational Radiobiology Group within the School of Cancer and Enabling Sciences at the University of

Manchester) and Adrian Harris (Weatherall Institute for Mollecullar Medicine at the University of Oxford)

has identified a set of characteristic splicing events in head and neck tumours that could predict for overall

survival.

Since alternative splicing involves removing sections of RNA before joining the remaining fragments

together into a mature mRNA molecule, different, and novel, splice variants can be found in sequencing

data by looking for reads that span the junctions between fragments. The application of direct sequencing

has the potential to give us a completely unbiased picture of all splicing events in a tissue or tumour. This

means that sequencing can be used to track how splicing patterns change between samples, allowing a

systematic exploration of both the functional and predictive consequences of these fine-grained changes

in protein expression.

We have been carrying out gene expression profiling studies in the Paterson Institute for several years now.

This work has been undertaken collaboratively between individual research groups, the MBCF and the ACBB

Group. The MCRC report in 2008 outlined two such studies that demonstrated the power of this technology

for identifying a) gene expression signatures associated with tumour hypoxia and b) for analysis of gene

expression in formalin-fixed paraffin-embedded (FFPE) material. Using the Affymetrix GeneChip® system

we can interrogate samples to determine which of the tens of thousands of human genes are active (or

inactive) in any tissue type. The ability to be able to profile gene expression in FFPE samples is particularly

relevant for clinical studies as large archives of FFPE material are available, and are especially important

Figure 2. Differentially expressed genes identified fromformalin fixed paraffin embedded (FFPE) tissue using

Affymetrix exon arrays. Each row corresponds to a mRNAtarget, each column, a tumour sample. Data are coloured by

expression level. Using this technology we are able to identifystatistically significant changes in expression. Ongoing work is

exploring the potential of these transcripts as biomarkers.

39

Discussion so far has concentrated on the analysis of DNA and RNA, but within the MCRC we have very

powerful biological mass spectrometry capabilities, commonly referred to as proteomics. Although, as

described above, a variety of different protein products can be produced from a single gene due to

alternative splicing; once any protein is produced it can be modified in a wide variety of different ways, all

of which may markedly alter its activity, specificity or function. Within the Biological Mass Spectrometry

Facility at the Paterson Institute we can carry out a variety of different analyses ranging from straightforward

protein identification, global quantitative comparison of protein expression in different tumour or tissue

types through to detailed analysis of post-translational modifications, either of specific proteins or more

global analysis.

Arguably, all these techniques become most powerful when they are used together in repeated rounds of

experimentation, where novel genes and binding sites are discovered, cells are perturbed to disrupt these

loci, and further rounds of sequencing or expression analysis performed in order to investigate the changes

that result. When the ability to be able to investigate protein expression and modification is placed

alongside technologies such as HTS and microarrays, it becomes possible to generate integrated views of

gene expression that encompass DNA, RNA and protein. A major goal therefore is to provide the close

cooperation and experimental integration required to support these complex, multi-platform experiments.

Within the MCRC we have access to a range of cutting edge technologies that will find increasing

application in the analysis of tumour initiation, progression, prognosis and response to therapies. In the

future the application of exomics - the analysis of all expressed sequences in many different tumours - will

provide a valuable picture of potential targets for therapy, and ultimately will help us to target genes and

their products selectively, eventually leading to the Holy Grail of personalised medicine.


38


Exactly how this is done depends on the platform, but with the majority of technologies, a single machine

run can generate many millions of relatively short (10s-100s of nucleotides) sequences, known as reads.

The volume of information that results from this places significant demands on the downstream computing

resources needed to analyse and store HTS experiments, and impacts both on the computing hardware

needed simply to manage the data, and the computational biology required to provide access to

appropriately detailed genome annotation and robust statistics (Figure 3). The ACBB Group at the Paterson

Institute is a highly collaborative mix of scientists from both the numeric- and the biological-sciences. The

majority of the group’s work involves collaborations with both basic and translational scientists within the

MCRC, and it is developing a variety of approaches for analysing HTS data.

Unlike other genome-wide technologies such as microarrays, RNA-Seq has the potential to offer true

nucleotide-level precision whilst generating profiles across the entire genome. This is important because

even with rapid progress in sequencing and analysis, we have yet to fully characterise the human genome.

In part this is because only about 2% of the 3 billion residues in the genome actually code for proteins - the

rest have, until recently, been considered to be relatively inert. However, using technologies including

sequencing and microarrays, many groups have been able to show that the majority of the genome is

expressed, at least at some stage, in some cells. This has led to an increased focus on the 98% of the genome

that does not code for proteins, and has revealed whole new classes of regulatory RNAs that, whilst they do

not originate from protein coding genes, are functional in their own right. RNA-Seq offers a remarkably

straightforward way to identify these genes, and then to explore how their pattern of expression changes

across, for example, normal healthy tissue and tumour cells. The ACBB Group is collaborating with a number

of groups in the search for novel non-coding RNAs with relevance to cancer, much of it driven by the

emerging regulatory role of these molecules.

As well as studying the expression patterns of different genes and splice variants, it is also important to

understand the processes that control these changes. Transcription factors are sets of proteins that bind

to DNA and help regulate the expression of sets of genes. Knowing which transcription factors are bound

to the regulatory region of a given gene can is therefore a key factor when developing an understanding of

the factors that govern its behaviour. ChIP-Seq is a technique in which individual transcription factors are

purified from a cell extract along with the section of DNA to which they are bound. This DNA can then be

released from the protein, sequenced, and aligned to the genome, revealing the locations where the

transcription factor was bound.

Although not yet routine, HTS is already becoming a key platform within the MCRC. Since its strength arises

from its ability to perform a relatively generic task – the efficient sequencing of millions of short reads – it

has a wide variety of applications, and a corresponding potential to benefit the work of many different

groups within the MCRC. Figure 3. RNA-Seq data visualised in the X:Map genome browser developed at the Paterson Institute. In the toppanel, peaks correspond to the number of reads aligning to the genome at that point. Data are associated withannotation representing the location and structure of known genes. The bottom panels provide more detailedannotation, and hyperlinks to other genome annotation resources.

41

four research-dedicated PET cameras: preclinical high-resolution HIDAC PET

and Siemen’s InVeon PET/CT systems for animal studies, a PET/CT whole body

scanner and a high resolution brain camera, which is the only one of its kind

in the UK. Clinical researchers also have access to the University’s research-

dedicated 1.5T MR system at the Wellcome Trust Clinical Research Facility

(WTCRF) and the 3.0T MR system situated at Hope Hospital. In addition, many

research studies are conducted in The Christie clinical imaging facilities. For

animal studies, a 7T preclinical MR is located in the Stopford Building.

Development of the MCRC imaging strategy has been supported by provision

of new imaging equipment and support posts. The Siemen’s InVeon is a state-

of-the-art preclinical PET/CT and was commissioned for use in December 2010.

This will run alongside the HIDAC system, but gives researchers access to CT-

capability, a small animal imaging modality that was previously unavailable

within The University of Manchester. Similarly, the 7T preclinical MR scanner

at the Stopford Building has been upgraded to give internationally

competitive MR capability in preclinical research. For clinical studies a

research dedicated 1.5T whole body MRI scanner has been installed at the

WMIC. These infrastructure changes have occurred in parallel with the

recruitment of three MCRC-funded research associates to specifically drive

forward research in preclinical and clinical MRI and PET. The University has

further supported appointment of two additional research associates to

support development of novel PET-tracers for preclinical application and

analysis of PET data from cancer studies.

Preclinical Imaging Research

Exciting progress has been made with preclinical whole animal imaging. The

key focus of current research programmes is the evaluation of cell population

dynamics (in particular cell proliferation and cell death) and the tumour

microenvironment. PET-based tracers of prolif-eration (18-F-FLT) and

metabolism (18-F-FDG) are being utilised as biomarkers for targeted therapies

(Figures 1 and 2) and also in genetic models where growth arrest is triggered

Imaging Research at the Manchester Cancer Research Centre

40



Ensuring the MCRC develops and implements a plan that strategically

integrates imaging science across the spectrum of its research is an

important part of establishing the MCRC on an international stage.

Advanced imaging methods play a core role in cancer research, early drug

discovery and increasingly in clinical management. The last 18 months have

seen pivotal changes in Imaging Research at the MCRC with the

implementation of an extensive plan to exploit the expertise in imaging

research and technology of MCRC partner groups within Manchester. These

developments are being coordinated by the newly formed MCRC Imaging

Group.

Under the umbrella of the MCRC Imaging Group, three inter-linked research

themes have been identified and form the basis for strategic developments.

The first focuses on research within imaging science itself, particularly the

development and validation of imaging biomarkers. This grows on the

established strengths and international reputation of Manchester in this area

and is co-ordinated by the MCRC Imaging Group. The second theme is the

strategic and co-ordinated development of preclinical imaging which has

been bolstered by the appointment of Dr Kaye Williams as preclinical lead,

upgrading of dedicated facilities and of the formation of the PReclinical

Imaging Executive (PRIME) to facilitate the development and implementation

of research projects and oversee all modalities of preclinical imaging. The

third theme focuses on translational clinical research with the establishment

of a Translational Research Imaging Group (TRIG). This group will drive

collaborative research and include clinicians and radiobiologists at The

Christie NHS Foundation Trust, the North West Medical Physics team, and

researchers at The University of Manchester.

The imaging research group already has access to high quality, well

established imaging facilities. The Wolfson Molecular Imaging Centre

(WMIC), situated on The Christie campus has its own cyclotron and dedicated

good manufacturing practice (GMP) radiochemistry facilities. The unit has

By Alan Jackson and Kaye Williams

Figure 1. Static PET images of mice with coloniccarcinoma heterografts showing differential

uptake of FDG (reflecting metabolism) and FLT(reflecting cellular proliferation).

Figure 2. Metastatic lung diseasedemonstrated by uptake of FDG (left). Images

on the right show the protective effect of thebioreductive agent NLCQ-1. Metastases can be

demonstrated using PET before anydetrimental effects on the animal are observed

(Cawthorne et al, Br J Cancer, 103, 201-208,2010).

biomarkers can expand. The activity of the TRIG has led to active

projects in brain, lung, breast, ovarian, cervical, colorectal, pancreatic,

renal, prostate, bladder, lymphoproliferative and hepatobiliary

cancers. Many of the studies are multimodality combining

advanced PET and MRI.

One example is an ongoing imaging-based study of Bevacizumab

(Avastin) in patients with metastatic colorectal cancer. This large

prospective clinical study includes over 70 patients each of whom

will have advanced MRI on five separate occasions. The study will

use qualitative biomarkers of the microvascular environment within

the tumour derived from dynamic contrast enhanced MRI. These

methods allow us to produce quantitative images of physiological

parameters including regional blood volume and endothelial

permeability surface area product. We have shown in a previous

study that these biomarkers are sensitive early indicators of

biological effectiveness with this agent (Figure 6). In the present

study we intend to test the hypothesis that early drug-induced

changes in these biomarkers will allow selection of patients who are

likely to show beneficial response. A subgroup of patients will also

have 18-FLT PET studies to measure the effects on tumour cell

proliferation. This powerful multimodality approach allows us to

examine the interactions between the primary anti-angiogenic

effect of the drug and the resultant change, if any, in proliferative

activity.

Another example is the increasing use of advanced PET ligands to

explore tumour biology in glioma. Manchester has a long history of

glioma imaging research based almost entirely around magnetic

resonance (Figure 7). Considerable biomarker development and

validation has occurred in this tumour group with a main focus on

biomarkers of the vascular micro-environment. Several new studies

are combining these advanced MR biomarkers with mechanistically

specific PET studies. These include the use of labelled methionine

(Figure 8) to identify tumour extent and recurrence, PK11195 (a

marker of microglial activation) to study the tumoural and peri-

tumoural inflammatory response in low grade glioma undergoing

dedifferentiation (Figure 9) and the use of 11C-verapamil to measure

the effect of PgP transporter blockade on drug uptake by

glioblastoma (Figure 10).

Imaging Biomarker Research

The development and validation of novel imaging biomarkers is the

core of a successful research imaging strategy. Imaging researchers

must be able to respond to the demands of the clinical and43


42


upon application of doxycyclin (Figure 3). In addition studies

are underway to validate biomarkers that reflect cell loss and

apoptosis using both PET (Figure 3) and MR imaging

modalities. In the latter case biomarkers derived from diffusion

weighted MRI are being evaluated as robust indicators of cell

death. These have been successfully applied in the context of

radiotherapy response (Figure 4) and are currently being

assessed in genetic models with controllable levels of

apoptosis. Collaborative studies with Dr Andrew Fagan (Centre

for Advance Medical Imaging, St. James’s Hospital Dublin,

Ireland) and Dr Friedrich Wetterling (Computer Assisted

Clinical Medicine, Medical Faculty Mannheim, University of

Heidelberg, Germany) have enabled the initiation of

preliminary multi-nuclear MRI studies (proton and sodium 23)

to complement the diffusion weighted imaging. In the case of

imaging the microenvironment oxygen-enhanced MR has

been applied as a novel MR technique to evaluate tumour

hypoxia (Figure 4) which is also being developed in the clinical

setting (Figure 11). Key to the further development of preclinical imaging in the MCRC is the developmental

of robust tumour models that reflect more accurately clinical disease. To support ongoing translational

research programmes, orthotopic models of glioma and colorectal liver metastases have been generated for

imaging and therapeutic studies within the MCRC (Figure 5).

To support the development of novel PET-tracers for preclinical application, a radiochemistry research

associate has been recruited to the research team. They will contribute to research within cancer and other

disease settings. One particular focus here is the exploitation of a novel technique that has been developed

within the WMIC to allow the 18-F-labelling of macromolecules (for example peptides, nucleic acids,

antibody fragments) for use as PET-tracers. This exciting development has been exemplified by labelling

therapeutic peptides and assessing bio-distribution and metabolism via PET for neuroscience applications.

In addition, the apoptotic tracer ML-10 is at the latter stages of validation for preclinical studies which will

be initiated imminently within the MCRC.

Clinical Imaging Research

Both The Christie NHS Foundation Trust and The University of Manchester have extensive portfolios of

clinical imaging projects and imaging-based clinical trials. The Translational Research Imaging Group (TRIG)

has been formed to coordinate and integrate these research portfolios. The clinical imaging research groups

within the individual MCRC members have strongly synergistic expertise providing considerable added

value to the strategic development of a research imaging portfolio. The TRIG provides access to expertise

not only in clinical and experimental CT, MRI and PET but also in advanced image analysis, systems

modelling, decision support, imaging trial design and implementation and integration of imaging research

into surgical and radiotherapy studies. The role of the TRIG is to ensure strategic development and growth

of clinical imaging research within the MCRC. In order to achieve these goals the TRIG is proactively

interacting with individual clinical research groups within the MCRC to identify areas where imaging

Figure 3. PET-based non-invasive imaging of proliferationand cell loss in genetic “switch” models. Tumour uptake of18F-FLT is an early response biomarker of inhibition ofproliferation (top panels) or activation of apoptosis(bottom panels). Models used express a dominantnegative inhibitor of oncogenic signalling (DN-PI3K; top)or constitutively activate caspase 3 (bottom) in responseto doxycycline treatment. Tracer uptake is maintained incontrol tumours in the same mice. Courtesy ofCawthorne, Smigova, Williams, Morrow, Simpson, Dive.

Figure 4. A) Oxygen enhanced MRI as a potentialbiomarker of hypoxia in tumours. The tumourregions that show a reduction in longitudinal

relaxation rate (R1) during oxygen versus airbreathing (blue regions) correlate with the extent

of tumour hypoxia (brown staining-pimonidazole). Courtesy of Linnik, Waterton and

Parker. B) Apparent diffusion coefficient as anearly response biomarker for radiotherapy (IR). IR

causes a significant increase in ADC within 72h oftherapy. Courtesy of O’Connor, Burke, Williams

and Waterton.

Figure 5. Development of robust preclinicalmodels of colorectal liver metastases and glioma

to support translational imaging andtherapeutic studies within the MCRC. 18F-FLT

uptake into colorectal liver metastases (A) andorthotopic gliomas (C) with comparative T1-weighted coronal MR images (B and D). Also

shown for the glioma is the T1-weighted imagepost-IV gadolinium contrast agent

administration (E). Courtesy of Pathmanaban,Babur, Gieling, Cawthorne, Smigova, Williams

and Bigger.

Figure 6. Changesin endothelial

permeability(images) and

fractional bloodvolume (graph) in a

colorectal livermetastasis treatedwith bevacizumab.

biological research communities by identifying and validating imaging

based biomarkers that are of direct relevance to the biological

mechanisms involved in disease behaviour or therapeutic interventions.

The last 10 years has seen a major focus on the development of imaging

biomarkers that describe the microvascular environment driven, almost

entirely, by the development of anti-angiogenic therapeutic agents. In

the coming years new biomarkers will be required which enable us to

measure novel therapeutic targets and mechanisms. These will include

biomarkers of local invasive behaviour, metastatic behaviour, apoptosis

and cell death. The MCRC Imaging Group is actively involved in the

development and validation of a wide range of novel biomarkers.

Examples include the development of imaging techniques which

measure soluble oxygen concentration for the investigation of hypoxia,

new measures to identify locally invasive behaviour at tumour margins,

novel methods for analysing heterogeneity within tumours from MRI

and PET and analytical techniques which allow the identification and

segmentation of tumour tissue from normal background tissue.

Hypoxia is a major area of research interest in oncology and yet there is

still no totally satisfactory imaging biomarker of tissue hypoxia. In part

this reflects the complexity of the hypoxic mechanism where areas of

well-perfused tissue may show clear cellular hypoxia. An ideal imaging

biomarker would demonstrate cellular hypoxia through a specific

molecular mechanism. Such tracers do exist for PET studies (18F-MISO,

ATSM) but they are not widely available, complex to use and each have

specific disadvantages relating to tracer distribution, severity of hypoxia

required to produce uptake and other factors. Simple measurements of

regional blood flow do not inevitably map to hypoxia and there is

significant effort taking place worldwide to identify a simple, cheap,

widely-applicable imaging biomarker of hypoxia. In Manchester we have

developed a technique to image the concentration of dissolved oxygen

in blood and interstitial tissue using the MR properties of oxygen

dissolved in water. We have already validated this technique in normal

tissues and tumours and are continuing to develop and validate it in a

number of studies (Figure 11).

Many groups of novel agents in development are expected to have a

direct effect on local tumour invasive behaviour and on metastatic

potential. There are remarkably few biomarkers of invasive behaviour

and we are attempting to develop and validate new approaches to

assessing this important hallmark of malignancy. One relatively simple but promising approach has been

to measure the gradients of signal intensity between the tumour and the surrounding normal tissue. We

have applied this technique in patients with glioblastoma where there is believed to be a separate, more

invasive phenotype. Figure 12 shows our technique for mapping the invasive profile of a tumour. This profile

45


clearly identifies invasive phenotypes associated with worse

prognosis. The analysis must be conducted on appropriate images

including images of free water diffusion, which is affected by tumour

invasion into surrounding white matter and images of fractional blood

volume, which are also specifically affected, by regional tumour

invasion. These methods are now being extended to tumours in

peripheral tissues.

18FLT is a widely used and potentially valuable biomarker of cellular

proliferation. Unfortunately, it is metabolised and cleared by normal

liver tissue so that uptake within the normal liver is high. Traditionally,

this has prevented its use in studies of liver tumours or metastases.

Within the MCRC Imaging Group we have developed a segmentation

method, based on the pharmacokinetic characteristics of tumour and

normal liver tissue which enables us to separate normal from

malignant tissue in FLT studies (Figure 13). This development means

that we can now study proliferative activity within the liver

considerably extending our potential portfolio of techniques.

Advanced cancer imaging research has a clear and exciting future

within the MCRC. PRIME and TRIG provide a new and active forum for

researchers to explore the possibility of imaging based studies in their

area. The imaging research community in Manchester is vibrant and

increasingly successful and we are encouraging all researchers within

the MCRC to take advantage of these facilities.

18FLT is a widely used and potentially valuable biomarker of cellular

proliferation. Unfortunately, it is metabolised and cleared by normal

liver tissue so that uptake within the normal liver is high. Traditionally,

this has prevented its use in studies of liver tumours or metastases.

Within the MCRC Imaging Group we have developed a segmentation

method, based on the pharmacokinetic characteristics of tumour and

normal liver tissue which enables us to separate normal from

malignant tissue in FLT studies (Figure 9). This development means

that we can now study proliferative activity within the liver

considerably extending our potential portfolio of techniques.

Advanced cancer imaging research has a clear and exciting future

within the MCRC. PRIME and TRIG provide a new and active forum for

researchers to explore the possibility of imaging based studies in their

area. The imaging research community in Manchester is vibrant and

increasingly successful and we are encouraging all researchers within

the MCRC to take advantage of these facilities.

44


Figure 7. Quantitative biomarker images of alarge glioblastoma. The image on the left

shows the fractional blood volume within thetumour and in the large feeding vessels. The

image on the right shows a biomarker ofreduced perfusion pressure developed inManchester. This indicates the areas ofreduced blood flow, hypoxia and active

angiogenesis within the tumour.

Figure 8. Images of methionine uptake (top)and contrast enhanced MRI in a patient

with a recurrent low-grade brain tumour.Note the high methionine uptake despite

the relative absence of contrastenhancement on MR.

Figure 9. A fused MR and PK11195 PET imagedemonstrating the distribution of activated

microglial cells within and around a low-grade glioma.

Figure 10. Axial and coronal sectionsthrough region HG1 (a) and HG2 (e) on T1-weighted contrast-enhanced MR imaging

showing tissue segmentation in (b) and (f).GM=red, WM=green, LG=purple, HG1=lightblue, HG2=yellow. VPM K1 maps before andafter TQD are shown centred on HG1 (c and

d), HG2 (g and h).

Figure 11. Two tumours (A and B) imaged usingdissolved oxygen (left), dynamic contrast

enhanced MR (centre) and analysed to show theproportion of perfused tissue (right). Note that

high levels of oxygen transfer are seen in tumourB in areas that are relatively avascular and

poorly perfused. Other areas show extremelylow oxygen transfer despite high perfusion.

Figure 12. The location of a brain tumour (top).The middle two images show the tumour

boundary with measurement lines at every pointallowing the automated calculation of the

tumour invasion profile. The image on the rightshows the distribution of these measurements

demonstrating clear areas of increased (red)invasion and areas of increased infiltration intoperipheral tissues (blue). These measurementscan then be projected on to the source images

(bottom).

Figure 13a & 13b. CT (9A) and FLT (9B) images ina patient with hepatic metastases. On the left-

hand side of B are the source FLT uptake images.Tumour cannot be differentiated from normalliver. On the right are filtered images from the

same dataset using a new technique developedin Manchester to show normal liver (top) and

tumour tissue (bottom).

47

Epidemiology of excess weight and cancer

In collaboration with the University of Bern, Switzerland, the group published

a seminal paper in the Lancet in 2008, quantifying the associations between

increased excess body weight and the risk of several adult cancers. This work

specifically showed that associations are: (i) sex-specific; (ii) common across

populations, including those from Asia-Pacific; and (iii) present for less

common malignancies (such as lymphoma, multiple myeloma, melanoma)

as well as the previously recognised common malignancies (such as post-

menopausal breast, colorectal, and endometrial). In collaboration with

Professor Iain Buchan, at the North West Institute for Bio-Health Informatics

(NIBHI), The University of Manchester, and investigators from the University

of Erasmus, Rotterdam, the group went on to use attributable risk approaches

and probabilistic modelling to show that excess body weight accounts for

Obesity and Cancer

46

Obesity and Cancer


Over the past five years, there has been a fundamental shift in the way the

scientific community investigates lifestyle and dietary aetiological factors for

common adult cancers; away from a ‘reductive approach’, for example dietary

micronutrients, to a more holistic ‘macro approach’ such as obesity. The

Obesity and Cancer Research Group has been at the forefront in quantifying

the association between body mass index (BMI) and risk of several cancer

types, and predicting that obesity may become a greater attributable risk

factor than smoking for new cancer cases in Europe over the next decade.

Evidence is also emerging that obesity is an adverse prognostic factor in

patients with cancer and a predictive lifestyle ‘biomarker’ of adverse

treatment response. To better understand the complex relationships

between obesity and cancer, the group is developing laboratory models along

two lines: chronic insulin exposure in in vitro cell models (hyperinsulinaemia

is a consistent feature in obesity); and in vivo models of human tumour

growth and progression in diet-induced and genetically-induced obese

animals.

Body mass index (BMI: expressed as kg/m2) is the standard method of

approximating body adiposity at a population level. Normal weight is defined

as a BMI between 18.5 and 24.9 kg/m2; overweight as 25.0 to 29.9 kg/m2; and

obesity as a BMI value greater than 30.0 kg/m2. Excess body weight is

overweight and/or obesity. The Obesity and Cancer Research Group

commenced in 2007, led by Andrew Renehan and based in the School of

Cancer & Enabling Sciences at the capital University of Manchester. The

group has parallel clinical and laboratory research activities. An underlying

principle to the research programme is the establishment of robust clinical

observations prior to developing human-mimicking models in the laboratory

(Figure 1). The primary cancer of interest is colorectal. The laboratory activities

are based in the Paterson Institute for Cancer Research, and integrated into

the Clinical and Experimental Pharmacology Group.

By Andrew G Renehan

Large bowel

Intestinal crypt

Obesity in apopulation

Cellularmodels

Figure 1. Cartoon illustrating the principle of taking robust clinical observations tolaboratory models – the ‘population to pipette’ approach.

124,000 new cancer cases in Europe per year. Evidence is also emerging that obesity is an adverse prognostic

factor in patients with cancer, and a predictive lifestyle ‘biomarker’ of adverse treatment response. The

group is currently exploring clinical databases to determine the impact of excess weight on anti-cancer

treatment responses and outcomes.

In vitro experiments – chronic insulin exposure

A variety of candidate biological mechanisms linking obesity with cancer risk and progression have been

suggested. Key among these is the insulin and insulin-like growth factor (IGF) system, and this is a focus in

the laboratory research programme. The acute effects of insulin and IGF-I on cancer cells are well known

as these growth factors increase cell growth, promote cell survival, and increase cell motility, attributes

which favour tumour progression. The effect of chronic insulin exposure on insulin-sensitive tissues, such

as fat and muscle cells, is well characterised in the diabetes and metabolic literature, but there is a paucity

of data on equivalent effects in cancer cells. Chronic insulin exposure is associated with adaptation of the

immediate downstream complex, insulin-receptor substrate (IRS)-1 and -2; specifically, there is decreased

tyrosine phosphorylation (the stimulatory arm) of IRS-1 and increased serine phosphorylation (the inhibitory

arm) of the IRS-1, with ultimate reduction in IRS-1 expression (Figure 2). Our early studies show, for the first

time, that in many cancer cell types, this adaptive mechanism to chronic insulin exposure is lost. In turn,

this led us to hypothesise that this failure to ‘take the breaks off’ insulin and IGF-I signalling may account

for why hyperinsulinaemia and some insulin treatments are associated with increased cancer risk and

progression.

We are currently exploring whether the lack of molecular adaptation among cancer cell types is determined

by mutations in downstream signalling, such as the KRAS and BRAF genes. These are key predictive

biomarkers for new therapies in colorectal cancer and, in turn, obesity and hyperinsulinaemia may be

modifiers of this predictive process.

49

In vivo models of obesity and cancer

Obesity is a complex multi-organ problem and to better understand its influence of cancer risk, progression

and treatment interaction, it is necessary to develop human mimicking animal models. This is not a

straightforward challenge. Thus, for example, the usual manner to develop a diet-induced obese laboratory

animal is through excess fat context in the diet. However, this approach produces only a modest phenotype

of excess insulin and it is not possible to disentangle the effects on tumours of excess dietary fat versus

increased body adiposity per se. Accordingly, we have chosen to use genetically induced obese murine

models where animals are hyperphagic i.e. they eat the same proportions of fat but excess calorie intake

compared with lean counterparts.

Early experiments show that the effects of excess insulin may differ for normal intestinal tissues compared

with neoplastic tissue; an observation consistent with the findings in the in vitro cell systems described

above.

Cancer Prevention Research Network

The Cancer Prevention Research Network (Chairs: Andrew Renehan and Anthony Howell) is a research

network within the Institute for Health Sciences at The University of Manchester. It offers an example of

the Manchester Academic Health Science Centre (MAHSC) in action bringing together disparate disciplines

from across academic teams in Manchester. Two examples in the field of obesity and cancer illustrate the

benefit of this network. First, the obesity and cancer group have supported the prevention team of the

Manchester Breast Centre (leads: Anthony Howell and Michelle Harvie) to optimise the role of surrogate

markers, such as serum IGF-I, in intervention weight-loss trials in women at high risk of breast cancer.

Second, in a collaboration with the Gynaecological Oncology team (lead: Henry Kitchener) and the MRC

Obesity and Cancer

48


InsulinIGF-I

IR

PI3K

AKT

Gene expression/Invasion

BAD

Bcl-2

Survival Gene expression/Proliferation

Hypoxia/ treatment resistanceTranscription

PAK-1 KRAS BRAF

ERK

mTor

IRS-1IRS-1

P302

P307

P612

P636/39

P1101

P 895

P 989

P 1229

tyrosine

serine

Figure 2. Schematic diagram of principle pathways linking insulin and cancer cell biological endpoints. Insert: phosphorylation andadaptation by the IRS-1 complex: insulin resistance is characterised by increased serine phosphorylations.

51

Obesity and Cancer

50


Clinical Trials Unit, the Obesity and Cancer Group are currently undertaking secondary analyses of the effects

of BMI on treatment outcome in women with endometrial cancer treated with the ASPAC trial.

Manchester Birmingham Biostatistics and Biomarkers Collaboration

Biomarkers are increasingly important tools in oncology and, in particular, to facilitate drug development.

The increased integration of these biomarkers into appropriately designed clinical trials is guiding the new

era of personalised therapeutics. For pharmacological and predictive biomarkers, desirable statistical

performance characteristics parallel those for diagnostic tests, such as high sensitivity, specificity and

accuracy. Such qualities are seldom achieved using a single serum biomarker, and thus the combination of

multiple biomarkers to develop a “composite serum biomarker”, with enhanced performance and event

classification is appealing. However, as the number of biomarkers increases, in parallel, there is an increase

in the complexity of statistical handling of data.

To address the needs for robust statistical frameworks to underpin future biomarker-driven trials, the

Clinical and Experimental Pharmacology Group (leads: Caroline Dive and Malcolm Ranson) in collaboration

with the Tumour Angiogenesis Group (lead: Gordon Jayson), jointly one of the largest research groups in

Europe developing biomarker-linked clinical oncology trials, has formed a collaboration with the

Birmingham Cancer Research UK Clinical Trials group (leads: Cindy Billingham and Philip Johnson). The

Birmingham unit has recently become a Medical Research Council (MRC) methodology hub. The statistical

team includes over twenty statisticians and clinical trial researchers. New posts appointed through the

MRC methodology hub scheme, and a two-city bio-statistical post, will contribute dedicated time to the

proposed collaboration.

The collaboration (abbreviated to MBBBC) has been headed by Andrew Renehan and supported by the

Manchester Cancer Research Centre. The first workshop was held in Manchester in February 2010; a second

workshop is planned for Birmingham in June 2010. The first workshop identified that the initial focus of

research will be the statistical handling of multiple blood-borne biomarkers. Future activities will include:

sharing of trial data (Figure 3); development of models to handle longitudinal data, for example, mixed-

effect models, time series modelling; and models to best classify disease events using composite

biomarkers, for example, artificial neural networks.

Time (days)

Figure 3. An example of complex biomarker data sampled longitudinally in a trial of 45 patients undergoing first-line treatment for metastaticcolorectal cancer (The SCOUT trial: data courtesy of Dr Mark Saunders). Each dot represents a sample measurement. Each patient has multiple

repeated measurements, which in turn, are correlated with each other. Some patients died during the time period of the trial (D).

53

The physiological importance of the MAPK cascades has been mostly

demonstrated by embryonic death caused by their functional deletion in mice.

Consistently, studies using cells derived from these genetically modified

animals have provided evidence that MAPK modules regulate numerous

cellular functions in response to distinct extracellular stimuli. For example, in

my previous studies, we have demonstrated that JNK is mainly activated by

cytokines and extracellular stresses. Like other MAPKs, JNK is activated upon

phosphorylation at Thr and Tyr residues by two MAPK kinases, MKK4 and

MKK7. Similar to the early embryonic death caused by the targeted deletion of

the jnk genes, the mkk4-/- and mkk7-/- mice die before birth. The non

redundant function of MKK4 and MKK7 in vivo may be due to their distinct

biochemical properties. Whereas MKK4 can also activate p38 MAPK, MKK7

functions as a specific activator of JNK.

Three genes, jnk1, jnk2, and jnk3 encoding 10 JNK isoforms have been identified

and implicated in promoting neuronal apoptosis during the development of

the central nervous system and in response to brain injury. The characteristic

morphological changes displayed by apoptotic cells reflect the activation of a

tightly regulated intrinsic cell signalling machinery that leads to the activation

of caspases. The ability of JNK to trigger the mitochondrial release of

cytochrome c via post-tanslational modifications of the pro-apoptotic Bcl-2

family members constitutes one mechanism by which JNK activates the

caspase cascade. However, in contrast to other cell types, redistribution of

cytochrome c is not the only regulated step during neuronal apoptosis. De

novo transcription via JNK-dependent regulation of AP-1 activity may

contribute to the additional changes that make a neurone competent to

receive the cytochrome c signal. This is supported by the similarity of the

phenotypic defect displayed by mice carrying a mutated c-jun gene in which

Ser63 and Ser73 residues known to be phosphorylated by JNK are converted

to Ala residues and the jnk3-/- mice.

Role of MKK4 in Ras-induced cancers

52

Role of MKK4 in Ras-inducedcancers


Among the signalling pathways strongly suspected to be involved in

mediating the effect of oncogenes in vitro is the mitogen-activated protein

kinase (MAPK) kinase 4 (MKK4), a non-redundant component of stress

activated MAPK signalling modules. However, the function of MKK4 in

tumourigenesis remains controversial with some studies indicating that

MKK4 is a tumour suppressor, while others have reported a pro-oncogenic

role of MKK4. To clarify these discrepancies, we have examined the role of

MKK4 in the development of skin tumours that occurs in situ in the mouse.

In contrast to the previous studies, this system recapitulates the complex

interactions between tumours and host as well as the cellular heterogeneity

of tumours. This is critical considering that the environment greatly

influences the spatial and temporal regulation of signalling pathways

activated by oncogenes, and consequently determines the biological outcome

of the response. Our results demonstrate that skin-specific MKK4-deficient

mice are resistant to tumourigenesis associated with oncogenic activation

of the ras gene.

Cells in every organism have the ability to respond to signals from their

environment, thanks to clever systems in which external stimuli trigger a

cascade of events which leads to a change in cellular behaviour. These

systems are known as cell signalling pathways and one of them, the mitogen-

activated protein kinase (MAPK) pathway, has been the focus of my group.

The MAPK family comprises three members: extracellular signal-regulated

protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK, also known as

stress-activated protein kinase, SAPK), and p38 MAPK (also known as cytokine-

suppressive anti-inflammatory drug-binding protein). Two main mechanisms

have been proposed to ensure specific transmission of the signals from

upstream kinases to MAPKs: i) scaffold proteins that assemble the different

components of a cascade; ii) physical interactions between the components

of a cascade. Both mechanisms may operate in parallel and allow different

responses of the same MAPK pathways to different stimuli.

By Cathy Tournier

However, apoptosis does not represent the only functional consequence of JNK activation. For example,

JNK appears to contribute to cell survival, cell proliferation, the immune response, and to mediate the effect

of certain oncogenes, including those associated with over-expression of mutant forms of ras. Ras belongs

to a large family of small GTPases that bind GTP and hydrolyse it to GDP. Constitutive active ras mutants have

been identified in cancers of many different origins, including pancreas (90%), colon (50%), lung (30%),

thyroid (50%), bladder (6%), ovarian (15%), breast, skin, liver, kidney, and some leukaemia. Site directed

mutagenesis have provided evidence that c-Jun is essential for transformation of fibroblasts by activated Ras

proteins. However, despite of its predominant role in controlling the transcriptional activity of c-Jun and c-

Jun expression, the requirement of JNK in Ras-mediated tumour initiation, progression and metastasis is not

rigorously established because of controversial findings.

Like JNK, the role of MKK4 in cancer appears complex with some data suggesting that MKK4 acts as a

tumour and metastasis suppressor, while others pointing to a pro-oncogenic role for MKK4. These

conflicting results have been an obstacle for considering MKK4 a valuable drug targets for cancer therapy.

To clarify these discrepancies, we have examined the effect of mkk4 gene deletion on the development of

skin tumours. The mice were treated with the carcinogen 7, 12-dimethyl-benzanthracene (DMBA) and with

the tumour promoter 12-O-tetradecanoylphorbol

13-acetate (TPA). This simple, versatile and highly

reproducible chemical carcinogenesis protocol

recapitulates the fundamental concept that

tumour development is a multi-step process that

includes: initiation of benign papilloma (mutation

of H-ras following DMBA treatment), promotion (a

clonal expansion of the initated cell triggered by

the repeated treatment with TPA to form a benign

tumour), and progression to malignant carcinoma

(an additional evolutionary step). Using this

model, we have discovered that the number of

tumours per mouse was greatly decreased in mice

lacking MKK4. These results provided the first

genetic demonstration that signalling

downstream of MKK4 is essential for tumour

formation in the skin (Fig. 1).

Based on these data, Cancer Research UK has

awarded us a project grant to specifically define

the role of MKK4 in tumour initiation and

progression, under normal conditions and in

response to anticancer therapies. In particular, we

will determine whether MKK4 is involved in the

development of chemoresistance by promoting

the expression of membrane drug transporters.

Over-expression of membrane drug transporters

such as P-glycoproteins (P-gls) and multidrug

55

resistance proteins (MRPs) constitutes one mechanism by which tumour cells can acquire resistance to

anticancer agents. This represents a substantial obstacle for the successful treatment of certain human

cancers. JNK has been implicated in increasing MRP1 expression. Furthermore, a recent study has shown that

xenografts of MKK4-deficient colon cancer cell lines exhibited greater sensitivity to anticancer agents. This

work is being developed using the chemically induced skin cancer model. We will translate these findings

to another epithelial cancer model: pancreatic cancer. The role of MKK4 in pancreatic cancer is highly

controversial with some studies presenting evidence that MKK4 acts as a tumour promoter while others

suggesting that it is a tumour suppressor. However, none of these studies have used models that accurately

recapitulate the features of premalignant or malignant ductal lesions. We propose to address this issue

using a mouse model which spontaneously develops the full spectrum of human pancreatic intraepithelial

neoplasias (PanINs) following the physiological expression of K-rasG12D in the pancreata. K-rasG12D is a

constitutive active mutant form of K-ras found in human pancreatic cancer. Some of these lesions progress

to invasive and metastatic adenocarcinomas.

However, whether pharmacological inhibition of MKK4 will reproduce the effect of mkk4 gene deletion

remains to be established before the therapeutic implication of inhibiting MKK4 activity for treating human

cancer becomes clear. This key question, namely to explore MKK4 as a potential anticancer drug target, is

being addressed in collaboration with the Drug Discovery Unit at the Paterson Institute for Cancer Research

who has identified a number of compounds that constitute good candidates for inhibiting MKK4 activity.

These compounds will be synthesised and tested in skin and pancreatic cancer cell lines carrying oncogenic

mutation in H-ras or K-ras genes, for their ability to i) inhibit JNK activity, ii) block cell proliferation and iii)

sensitise cells to the apoptotic effect of genotoxic drugs (e.g., doxorubicin). The specificity of the compounds

will be established by comparing their effect to that associated with the down-regulation of MKK4

expression using siRNA technology. The next step will be to test the effect of the compounds on tumour

growth in our mouse models of skin and pancreatic cancer. One potential important outcome of our studies

is the validation of MKK4 as a drug target for the treatment of cancers. In particular, combined with

conventional DNA-damaging chemotherapeutics, inhibitors of MKK4 may provide a new strategy to

overcome resistance of tumours to the current therapies.

Role of MKK4 in Ras-induced cancers

54


DMBA/TPA

MKK4/MKK7

JNK

c-Junp53

p21 EGFR

APOPTOSIS

CELL CYCLEARREST

PROLIFERATION

Ras

Figure 1. Schematic role of MKK4 in skin cancer. Our results show that MKK4has a pro-oncogenic function in the skin. The mechanism by which MKK4promotes tumourigenesis in the skin appears to be mediated via JNK/c-Junsignalling. Downstream targets of c-Jun include the epidermal growth factorreceptor (EGFR). The ability of EGFR to prevent cell differentiation, therebymaintaining keratinocytes in a proliferative state, is one mechanism bywhich c-Jun contributes to tumour formation upon Ras activation. Inaddition, MKK4 may be involved in malignancy by decreasing p53 stability,and thereby down regulating p21 expression, via JNK independently of c-Jun.This model is supported by evidence that the loss of p53 enhances malignantprogression of chemically induced skin tumours.

57

research, the Patient Treatment Centre will provide improved facilities for

service chemotherapy delivery, hospital pharmacy and aseptics and a new

private patient facility.

Phase I trials are experimental studies that seek to define the safety and

pharmacology of new treatments in man and they represent the foundation

stone for future therapeutic developments. Each trial involves the detailed

study of a relatively small number of cancer patients who have usually

received, and have previously failed, established therapies for their tumour.

Each patient is carefully evaluated and detailed clinical observations are

complemented by translational research. Pharmacokinetic studies investigate

how the body distributes and excretes the new agent and pharmacodynamic

studies examine the effects of the new agent on tumour and normal tissues.

The first opportunity to evaluate human pharmacodynamics and to study

proof of mechanism and proof of principle occurs in these Phase I trials.

Although challenging, early phase research helps to define potential

biomarkers to assist in dose optimisation, improve patient selection and

develops the logistics required for future translational research in larger

patient cohorts. Phase I studies are followed by statistically more amendable

phase II trials, where larger cohorts of patients with a single tumour type are

evaluated. The need for biomarkers to allow clinicians to stratify patients and

to optimise treatment has become more important as the molecular

characteristics of tumours has become more clearly defined, and as

mechanism based treatments have been developed. Providing personalised

cancer medicine represents an important objective for improving cancer

patient survival in the future. The recent demonstration of epidermal growth

factor receptor (EGFR) mutations, which define a highly sensitive sub-

population of non small cell lung cancer patients for EGFR inhibitors, the

predictive potential of KRAS mutations in clinical decision making for EGFR

monoclonal antibody therapy in colorectal cancer and the development of

The New Patient Treatment Centre

56



One of the research areas within the MCRC with an international reputation

is early phase clinical trials and the closely aligned translational research. The

original development and opening of The Christie’s Derek Crowther Unit

(DCU) in 2003 was an important milestone in developing world class early

phase clinical research in Manchester. Since its original opening, the DCU

has flourished, doubling its research activity and patient recruitment, but in

the process becoming too small to support its research portfolio. Over the

last few years Manchester has become one of the most active early phase

clinical trial centres in Europe. During the last three years The Christie has

enrolled over 3,900 patients to 70 Phase I trials, 132 Phase II trials and 44

biomarker studies. During the last five years Manchester’s early phase clinical

research activities have been strengthened by the development of the Clinical

and Experimental Pharmacology Group and of GCLP-compliant research

laboratories in the Paterson Institute for Cancer Research, along with opening

of the Wolfson Medical Imaging Centre (WMIC), the MCRC Biobank and the

Cancer Research UK Drug Discovery Centre. A successful Clinical Research

Infrastructure Award from the CR-UK/Department of Health (DoH)/Wolfson,

coupled with substantial additional investment from The Christie and other

partners, has now enabled the MCRC to enhance the clinical infrastructure

support for early phase clinical research in Manchester.

Detailed proposals for the new Patient Treatment Centre received approval

from The Christie NHS Foundation Trust Board in late 2008, and local

planning approval was granted in December 2008. The original DCU building

was demolished in February 2009 and its research activity relocated to a

temporary building situated on the north side of The Christie site. Building

work on the £35M Patient Treatment Centre commenced in January 2009 and

was opened in November 2010. It will provide space for a doubling of research

activity and make Manchester one of the world’s largest centres for early

phase clinical trials. Alongside improved capacity and facilities for clinical

By Malcolm Ranson

59


58


BRCA mutation testing in the clinical development of PARP inhibitors in breast and ovarian cancers, are

recent examples of oncology moving towards personalised medicine. In keeping with this it will come as

no surprise that over 80% of the early phase trials conducted by the DCU include biological sample collection

(tissue, blood, urine, etc) and that the translational activities of the DCU have grown steadily since 2004.

The present DCU laboratory processes over 11,000 research samples a year and larger laboratory facilities to

support its growing translational research portfolio have been included in the new Patient Treatment Centre.

Opportunity has also been taken to co-locate this important development in the main treatment area of The

Christie (See Figure 1). This will allow researchers to extend translational science into later phase clinical

trials, an important step since phase II and phase III trials have the necessary statistical power for developing

decision making tools based upon biomarkers. Delivering the necessary high quality clinical science and

conducting it to good clinical practice (GCP) and good clinical laboratory practice (GCLP) involves close

multidisciplinary team working. Support from MCRC partners and from CR UK and DoH to the Manchester

Experimental Cancer Medicine Centre (ECMC) has helped us to respond to the changing research landscape.

This core support has funded key staff for our GCLP research laboratories and provided technical support

staff in pharmacy, radioimmunotherapy, imaging and radiobiology. To support the future growth of

translational research the MCRC has also recently invested in a Laboratory Information Management System

to cover the Paterson and DCU GCLP Laboratories. This will provide powerful linkage and management

tools for large clinical and research laboratory datasets.

As the research portfolios of the DCU and ECMC have grown, the world class biomedical imaging facilities

of the WMIC have become increasingly deployed to provide pharmacodynamic imaging information during

experimental treatment. Recently, a growing trend of cross-cutting research has emerged in which

advanced biomedical imaging is combined and compared with blood and tissue borne biomarkers. These

ground breaking studies should help optimise future patient treatment particularly for anti-angiogenic and

targeted agents. The WMIC currently leads over a dozen imaging protocols within the Manchester ECMC

portfolio and twenty further oncology studies are currently in discussion or set-up.

The pharmaceutical and biotech industry recognises that the UK is a good place to do complex early phase

clinical trials and, although the climate for clinical research in the UK has become more complex and

administratively challenging during the last five years, Manchester has responded to these challenges and

has seen substantial growth in its research activity and in patient recruitment. The Christie’s research and

development department has set itself challenging new targets for trial set-up times and for performance

management to help ensure that Manchester remains internationally competitive.

Historically there has been a dearth of well trained clinical and translational scientists in Europe. Training

of scientists and clinicians with expertise in early phase therapeutic development and in translational

science therefore continues to be an important area of investment. Currently our early phase trial activity

is heavily dependent upon a relatively small number of senior clinical researchers, and two principal

investigators currently account for over half our phase I trial recruitment. To realise the full potential of our

new infrastructure developments, additional senior clinical researchers engaged in early phase clinical

research will be recruited over the next five years. Since 2006, a highly successful Clinical Pharmacology

Fellowship scheme, funded by Cancer Research UK and AstraZeneca, has enabled eight promising young

clinical researchers to develop research skills in early phase clinical trials and experimental pharmacology

and to undertake PhD training. Complemented by training programmes for scientists in GCLP and an MRes

course in Translational Medicine, established by Professor Andrew Hughes, our investment in future talent

remains a key objective.

Figure 1. The new Patient Treatment Centre under construction in March 2010. The main entrance to The Christie is out of frame to theright, and the proximity of the patient treatment centre to many of the wards (to the right of the new build) can be clearly seen.

Chris Morrow, Kathryn Simpson, Luke Harrison and Tetyana Klymenko (Paterson Institute for Cancer Research) and Tom Owens

and Fiona Foster (Faculty of Life Sciences, The University of Manchester). - The Manchester Cancer Research Centre Conference

- Harnessing Apoptosis – page 10

Chris Morrow joined the Clinical and Experimental Pharmacology group at the Paterson

Institute for Cancer Research in April 2004 working as a postdoctoral research associate for

Professor Caroline Dive. Prior to this he read molecular biology at the University of

Edinburgh, gaining a BSc in 2000, before carrying out postgraduate research into cellular

mechanisms ensuring faithful chromosome segregation at The University of Manchester

under the supervision of Professor Stephen Taylor. He now works in the preclinical section

of Professor Dive’s laboratory, focusing on combinations of anti-cancer drugs which induce

cancer cell death and determining the underlying molecular mechanisms behind the drug

interactions.

Kathryn Simpson joined the Clinical and Experimental Pharmacology (CEP) group at the

Paterson Institute for Cancer Research in 2005 as a postdoc to work on small molecule

inhibitors of apoptosis, but also with a focus on developing the clinical and translational

research activities in Professor Caroline Dive’s laboratory. Kathryn became involved in the

implementation of a biomarker research project in CEP, utilising cutting-edge mass

spectrometry technologies, in close collaboration with the expertise in Professor Tony

Whetton’s Stem Cell and Leukaemia Proteomics Research Laboratory. The Clinical

Proteomics Team was soon established, led by Kathryn, in order to help fulfil an unmet

clinical need in cancer care by the discovery of novel biomarkers with the aim of identifying

predictive, prognostic, toxicity and/or response information from the circulating

complement of proteins in patient blood.

Luke Harrison completed a degree in Biochemistry at Newcastle University and

subsequently stayed in Newcastle to undertake a PhD in molecular pharmacology with

Professor Herbie Newell and Dr Mike Tilby. His PhD focused on studying the mechanistic

interactions and therapeutic uses of novel kinase inhibitors in combination with

conventional cytotoxic drugs. After completing his PhD Luke joined Professor Caroline

Dive's Clinical and Experimental Pharmacology group at the Paterson Institute for Cancer

Research in April 2008 as a postdoc where he continues his interest in preclinical

pharmacology. He is currently working on the effect of the tumour microenvironment on

the efficacy of apoptosis-inducing drugs.

61

Author Biographies

60

Author Biographies


Tetyana Klymenko joined the Clinical and Experimental Pharmacology Group in August

2008 as a postdoctoral scientist. She graduated from Kharkov State University in 1999

with her master thesis in biochemistry of viral RNA based on experimental work done at

the Puschino Institute of Protein Research, Russian Academy of Sciences. Following her

work on activation of transcription targets of HIV in Tampere, Finland, as a visiting scientist,

she completed a PhD on regulation of gene expression at the European Molecular Biology

Laboratory (EMBL) in Heidelberg, Germany.

Thomas Owens is in his third year of postdoctoral studies at The University of Manchester.

He gained a BSc in Biochemistry with Industrial Experience from The University of

Manchester in 2002. His placement year was at Evans Vaccines (now Novartis) in Liverpool,

where he worked in the Hepatitis-B-vaccine production facility. Following his degree he

worked on oligonucleotide synthesis at Applied Biosystems, before returning to

Manchester on the Wellcome Trust 4-Year PhD programme in 2003. His postgraduate

studies with Professor Charles Streuli examined the role that IAP proteins had in regulating

apoptosis in mammary epithelial cells. Since completing his PhD he continued to study the

mechanisms of apoptosis regulation with Dr Andrew Gilmore, focusing on the signals that

control the critical point in the life and death decision of a cell.

Fiona Foster is currently undertaking postdoctoral studies at The University of Manchester,

where she is investigating the role of focal adhesion kinase (FAK) in breast cancer initiation.

Fiona undertook a BMedSci at the University of Sydney, Australia, prior to obtaining her

PhD from the same institute in 2001. Fiona began her postdoctoral career in breast cancer

research at the University of Reading in the UK. This involved work on two projects, the first

examining the role of class II PI3 kinases and the second looking at the role of a protein

modification which could potentially make breast cancer cells immuno-suppressive. After

moving to The University of Manchester in 2006, Fiona has continued her research into

breast cancer, looking at the role of apoptosis suppression in breast cancer, first of all that

mediated by the Inhibitor of Apoptosis proteins and now that mediated by FAK.

63

Author Biographies

62


Fiona Blackhall trained in Manchester and for two years in Toronto with Dr Frances

Shepherd. She was appointed Consultant Medical Oncologist at the The Christie NHS

Foundation Trust in 2005, joining the Manchester Lung Group led by Professor Nick

Thatcher. She was awarded Honorary Senior Lecturer in the School of Cancer and Enabling

Sciences at The University of Manchester in 2007. She is an active clinical trialist with a

focus on development of mechanism based therapies and has a major role in biomarker

research, now leading the lung cancer focus group within the Clinical and Experimental

Pharmacology Group at the Paterson Institute for Cancer Research. She is Chair of the

translational subgroup of the recently established European Thoracic Oncology Platform,

a member of the NCRI Lung Clinical Studies Group and leads the MCRC lung cancer

research team.

Professor Caroline Dive is a Senior Group Leader at the Paterson Institute for Cancer

Research and Professor of Pharmacology in the School of Cancer and Imaging Sciences at

The University of Manchester. She is a co-director of the Clinical and Experimental

Pharmacology Group (CEP) within the Paterson Institute for Cancer Research. After

completing her PhD studies in Cambridge, Caroline moved to Aston University's School of

Pharmaceutical Sciences in Birmingham where she started her own group studying

mechanisms of drug induced tumour cell death. She then moved to what became the

Faculty of Life Sciences at The University of Manchester to continue this research and was

awarded a Lister Institute of Preventative Medicine Research Fellowship. Caroline joined

the Paterson Institute for Cancer Research in 2003 to set up the CEP, interfacing with the

Derek Crowther Unit for early clinical trials at The Christie. The CEP aims to drive early

clinical evaluation of novel mechanism-based targeted therapies by providing biomarkers

expertise, analysis of which acts as a readout of drug activity within the body.

Ivan Ahel - DNA Damage Response – page 20

Ivan Ahel obtained his BSc and MSc in Biology at the University of Zagreb, Croatia, in 1997

and 2000 respectively, before undertaking a PhD in Biology with thesis work carried out at

Yale University, USA, between 2000-2003. His undergraduate and PhD research experience

includes the regulation of the DNA damage response in actinomycetes and studies of the

mechanisms ensuring the fidelity of protein biosynthesis. In 2004 Ivan joined the Cancer

Research UK London Research Institute as a postdoctoral fellow investigating the

identification and characterisation of novel DNA repair proteins. In January 2009 he moved

to the Paterson Institute for Cancer Research as Group Leader for the DNA Damage

Response Group.

Tim Ward, Jian-Mei Hou, Matthew Krebs, Fiona Blackhall and Caroline Dive - Lung Cancer Circulating Tumour Cells as

Biomarkers and a Window to Metastasis Biology – page 16

Tim Ward is a translational scientist working within the Clinical and Experimental

Pharmacology Group at the Paterson Institute. He is the Pharmacodynamics Manager

responsible for the development, validation and implementation of all assays used by the

group in early phase clinical trials. He has over 30 years experience in cancer research,

principally anti-cancer drug development specialising in anticancer drug screening and

DNA damaging assays as well as extensive experience in cell culture assays. He currently

sits on the Cancer Research UK New Agents Committee (NAC). His current research

interests are centered on detecting and characterising circulating tumour cells and micro

emboli. He also has a keen interest in utilising circulating DNA as a biomarker of tumour

DNA to detect specific tumour mutations which impact on response to modern targeted

agents.

Jian-Mei Hou gained an MD degree through a seven year training program from West

China University of Medical Science in 2002. She subsequently spent three years studying

towards a PhD that focused on bio-immunotherapy of tumours at the National Key

Laboratory of Biotherapy, West China Hospital, Sichuan University. Jian-Mei spent a year

working on preclinical drug safety evaluation in National Chengdu Centre for Safety

Evaluation of Drugs. In May 2006, she joined the Clinical and Experimental Pharmacology

(CEP) Group at the Paterson Institute as a Cancer Research UK China Fellow. Her research

interests have since been focused on circulating tumour cells as a window into metastasis

biology in small cell lung cancer.

Matthew Krebs is a clinical research fellow within the Clinical and Experimental

Pharmacology Group at the Paterson Institute for Cancer Research. He completed his

degree in Medicine at The University of Leicester in 2001 and took opportunity to spend an

undergraduate elective period at the London Regional Cancer Centre, Ontario, Canada.

Following General Medical training in Manchester he commenced specialist training in

Medical Oncology at The Christie NHS Foundation Trust in 2005. Matthew subsequently

joined the Cancer Research UK/AstraZeneca PhD Clinical Fellowship programme in

November 2007. His research focuses on the isolation and characterisation of Circulating

Tumour Cells from patients with lung cancer with a view to developing these as predictive

and/or pharmacodynamic biomarkers in early phase clinical trials.

64

Manchester Cancer Research Centre Research Report Author Biographies

65

Anthony Howell is Professor of Medical Oncology and is the Director of the Breakthrough

Breast Cancer Research Unit, The University of Manchester. Formerly he was Chairman of

the UKCCCR, the British Breast Group and the Manchester Breast Centre, was the Research

& Development Director of The Christie NHS Foundation Trust and the Cancer Research

Network. His interests are the endocrine therapy and biology of the breast and breast

cancer with a particular interest in prevention. He has published over 500 papers mainly

in these areas.

Crispin Miller, Jenny Varley and Stuart Pepper - Cancer Genomics in the Era of High Throughput Sequencing – page 34

Crispin Miller studied Artificial Intelligence as an undergraduate before completing a PhD

in Computational Biology, which focused on the development of fast pattern matching

tools for nucleotide and protein sequence analysis. This was followed by an MRC training

fellowship in Bioinformatics. Crispin joined the Paterson Institute for Cancer Research in

2002, and currently heads the Applied Computational Biology and Bioinformatics Group.

Jenny Varley obtained her degree and PhD from the University of Leicester before

undertaking postdoctoral work in Geneva funded by a Royal Society Exchange Programme

Fellowship. After further postdoctoral work Jenny was appointed as a non-clinical lecturer

in Pathology at the University of Leicester, and subsequently moved to the Paterson

Institute in 1992. Jenny was head of the Cancer Genetics Group until 2001 and was

awarded the post of Honorary Professor of Experimental Oncology at The University of

Manchester in 2002. Jenny was appointed to the role of Assistant Director (Research) at

the Paterson Institute in 2001, and one of her major roles is developing and managing all

the scientific service units at the Institute.

Donald Ogilvie and Allan Jordan - Drug Discovery in the Manchester Cancer Research Centre – page 24

Donald Ogilvie heads the newly formed Drug Discovery Centre in the MCRC. He joined the

Paterson Institute as a senior group leader in February 2009 after a twenty year career in

the pharmaceutical industry. Donald obtained an MA in Biochemistry at Oxford in 1980

before working at the John Radcliffe Hospital for eight years on the role of proteases in

breast cancer then inherited connective tissue disorders. The latter was the basis of his

D.Phil degree. In 1988 he joined ICI which subsequently became Zeneca then AstraZeneca.

For most of his industrial career, Donald worked on cancer drug discovery and early clinical

development and he was directly responsible for the delivery of ten novel cancer

development compounds, several of which have progressed to Phase II & III clinical trials.

Allan Jordan joined the Drug Discovery Centre in July 2009 as Head of Chemistry. After

gaining a BSc in Chemistry from UMIST in 1993 and a short spell as a teaching assistant in

Arizona, he returned to UMIST to conduct post-graduate research into anticancer natural

products. After post-doctoral work at the University of Reading, he joined RiboTargets in

Cambridge (now Vernalis) where he worked on a number of therapeutic areas at various

stages of the research pipeline. Alongside involvement in a number of oncology

programmes, ultimately leading to the clinical evaluation of Hsp90 inhibitors in

conjunction with Novartis, he became involved in CNS research programmes where he was

a project leader on a GPCR drug discovery programme and was also involved in the

management of Vernalis’ clinical programme for Parkinson’s disease.

D Gareth Evans and Anthony Howell - Genetic Medicine and the Genesis Prevention Centre – page 28

D Gareth Evans has a national and international reputation in clinical and research aspects

of cancer genetics, particularly in neurofibromatosis and breast cancer, and is Chairman of

the NICE (National Institute for Clinical Excellence) Familial Breast Cancer Guideline

Development Group. He has developed a clinical service for cancer genetics in the North

West Region and lecture within the UK and internationally on hereditary breast cancer

and cancer syndromes. He has also developed a regional training programme for clinicians,

nurses and genetic associates in breast cancer genetics, and established a system for risk

assessment and counselling for breast cancer in Calman breast units.

66 6767

Author BiographiesManchester Cancer Research Centre Annual Research Report

Andrew G Renehan – Obesity and cancer – page 46

Andrew G Renehan is a Higher Education Funding Council for England (HEFCE) Senior

Lecturer in the School of Cancer and Imaging Sciences at The University of Manchester,

and Honorary Consultant in Colorectal and Pelvic Surgery at The Christie NHS Foundation

Trust. He heads the Colorectal Cancer and Obesity subgroup within the Clinical and

Experimental Pharmacology Group at the Paterson Institute for Cancer Research, and is

also Chair of the Cancer Prevention Research Network.

Cathy Tournier – Role of MKK4 in Ras-induced cancers – page 52

Cathy Tournier completed her PhD in 1996 and subsequently joined the laboratory of

Professor Roger J Davis (Howard Hughes Medical Institute, University of Massachusetts,

USA) to pursue her training in the field of research on the mitogen-activated protein kinase

(MAPK) signalling pathways. In July 2000, she was appointed as a lecturer in the Faculty of

Life Sciences at the University of Manchester. A year later, she was awarded a Senior

Research Fellowship from the Lister Institute of Preventive Medicine to focus on her

research. Cathy currently leads the Gene Expression and Cell Signalling Group.

Malcolm Ranson – The New Patient Treatment Centre – page 58

Malcolm Ranson completed his PhD in Manchester after obtaining medical and

Pharmacology degrees. Subsequently he was awarded an EORTC/NCI scholarship at the

National Cancer Institute, USA. He is currently Professor of Medical Oncology and

Pharmacology in the School of Cancer and Enabling Sciences at The University of

Manchester and heads the Manchester Experimental Cancer Medicine Centre. He is based

at The Christie where he is the Clinical Director of the Derek Crowther Trials Unit - one of

the largest early phase oncology clinical research units in Europe - and is co-Director of the

Clinical and Experimental Pharmacology Group within the Paterson Institute for Cancer

Research.

Stuart Pepper has run the Molecular Biology Core Facility for the Paterson Institute for

Cancer Research for the last eight years. The Core Facility provides a range of services

including DNA sequencing, DNA extraction and quantitative polymerase chain reaction

(PCR) analysis. The Core Facility also houses the Paterson’s Mass Spectrometry facility,

which provides state-of-the-art protein analysis services. During the last six years, Stuart

has also managed the Cancer Research UK GeneChip Microarray Facility, providing access

to cutting edge microarray technology for all Cancer Research UK grantees. The Core

Facility team have collaborated widely with local and national researchers and have

produced several publications using the latest microarray protocols available.

Alan Jackson and Kaye Williams - Imaging Research at the Manchester Cancer Research Centre – page 40

Alan Jackson joined The University of Manchester in 1993 and was appointed Professor of

Neuroradiology in the Imaging Sciences Group within the Faculty of Human and Medical

Sciences at The University of Manchester in 1995. Prior to this, he was a full-time NHS

clinical neuroradiologist at the Manchester Royal Infirmary. Alan holds honorary chairs in

Neuroimaging at the Universities of Liverpool and Bangor and is also an honorary clinical

consultant at the Salford Hospitals NHS Trust and the Manchester Royal Infirmary.

Kaye Williams received a BSc in Molecular Biology from The University of Manchester and

a PhD from the Paterson Institute for Cancer Research. Following Research Associate and

Research Fellow positions within the Experimental Oncology Group of the School of

Pharmacy and Pharmaceutical Sciences (headed by Professor Ian Stratford), she was

awarded the British Association for Cancer Research/AstraZeneca Frank Rose Young

Scientist Award in 2005. In January 2006 Kaye was appointed Senior Lecturer within the

School of Pharmacy. She currently heads the Hypoxia and Therapeutics Group and is the

MCRC lead for Preclinical Imaging.

Manchester Cancer Research CentreThe University of ManchesterWilmslow RoadManchesterM20 4BX

tel: +44 (0) 161 446 3156fax: +44 (0) 161 446 3109

www.manchester.ac.uk/mcrc

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