Oxford Cancer
Research Centre
Clinical Research
Training Fellowships
Projects 2013
INTRODUCTION
This handbook provides an overview to the Clinical Research Training Fellowship DPhil intake for the Oxford Cancer
Research Centre at the University of Oxford.
Please note that the handbook provides an extensive, yet not an exhaustive list of projects. We are happy to
consider additional projects that you may have developed in discussion with other researchers in the University,
where the supervisor demonstrates their agreement in supervising your project and sponsoring your application.
We advise prospective applicants to discuss their research interests with potential supervisors prior to submitting
an application form.
The programme is administered by the Oxford Cancer Research Centre at the Department of Oncology and all
enquiries should be made via the OCRC (email [email protected]).
SELECTION PROCESS OF FELLOWS
Applicants should demonstrate:
 a strong academic track record, e.g. a BSc (or equivalent) with a 1st or 2:1; honours or distinction in the MB
finals; evidence of any academic prizes;
 any previous research experience, which may include publication of research
 a strong commitment to the future pursuit of research
Additional criteria:
 It is envisaged that those appointed will have some specialist experience – typically having obtained MRCP or
equivalent, but not CCT or consultant status.
 The posts are open to those from the UK and also from abroad, but candidates must be eligible to undertake
clinical practice in the UK. Successful applicants will be required to hold an honorary clinical contract and therefore
must ensure that they have fulfilled all the criteria required of the General Medical Council and National Health
Trust. Please see the General Medical Council website for further details.
 Selection of fellows will be by interview and on the basis of academic ability, clinical competency, and a welldefined understanding of the proposed project.
HOW TO APPLY
All applicants must complete a short online application form and a CRF question form. To apply for this role, please
click on the ‘Apply Now’ button in the link below.
Available Projects are summarised on the following pages.
Timetable for admissions
The deadline for receipt of applications including all supporting materials is 12.00 noon on January 4th 2013.
Interviews are scheduled for the week of 28th January. The start date for successful candidates will ideally
be Michaelmas Term 2013 (October 2013).
Distinguishing oncogenic HIF pathways in Renal Cancer
Scientific Supervisor: Dr David Mole
Clinical Supervisors: Prof Peter Ratcliffe and Dr Andrew Protheroe
The aim of this project is to distinguish HIF transcriptional targets that are "driver" rather than "passenger" events in
renal cancer.
Project Proposal
Kidney cancer is increasing in incidence, with more than 8,500 cases / year in the UK. Without surgical cure,
prognosis remains poor, despite recent advances in medical management. The most common form of kidney cancer
is clear cell renal carcinoma (CCRC), the majority of which bear mutations in the von Hippel-Lindau tumour
suppressor (pVHL). A major function of pVHL is regulation of the transcriptional response to oxygen through targeted
degradation of hypoxia inducible factor (HIF) and evidence indicates a pathogenic role for this pathway in renal
cancer.
Inactivation of pVHL upregulates the HIF response, leading to enhanced transcription of many hundreds of genes
with both tumour promoting actions (e.g. angiogenesis, stem cell maintenance, metabolic adaptation, epithelial-tomessenchymal transitioning, invasion, metastasis, and resistance to radio- and chemotherapy) and antitumourigenic
actions (e.g. apoptosis) as well as tumour neutral activity. Activation of all of these genes in VHL defective CCRC will
occur in parallel and the project aims to distinguish "driver" from "passenger" HIF pathways using the following
aproaches.
(a) The first approach will use the concept of convergent evolution and the large degree of both inter- and intratumour heterogeneity in renal cancer, to determine key HIF targets by determining those that are common to many
tumours. HIF ChIP-seq will be undertaken in multiple CCRC cell lines and from multiple tumour samples and
combined with expression profiling to determine a core set of HIF targets that are conserved between renal tumours.
(b) A second approach will utilise the growing body of genetic evidence (both germline and somatic) to crossreference these genome-wide patterns of HIF-binding with pan-genomic signals of renal cancer predisposition and
determine those of particular relevance to pathogenesis of CCRC. We have previously used such an approach to
identify a long-range enhancer of the cell-cycle regulator, cyclin D1 that is important in renal cancer.
(c) In a third parallel approach, a defined set of CCRC HIF transcriptional targets will be examined for effects on
tumour growth using a high-throughput screening strategy. Essentially, pooled libraries of "bar-coded" shRNAs or
cDNAs will be expressed in CCRC cells in tumour xenografts. Next-generation sequencing will then be used to
measure the abundance of each shRNA or cDNA before and after growth in the xenograft. Cells containing an shRNA
or cDNA conferring a survival advantage will outgrow the others and these sequences will be enriched during tumour
growth.
Clinical/Translational relevance of project
Current medical therapies in kidney cancer already centre on specific aspects of the HIF response, namely mTOR
inhibitors, VEGF receptor tyrosine kinase inhibitors and anti-VEGF antibodies. However, these are of limited efficacy
and there is therefore a need to identify new treatment strategies. Identifying "driver" genes and pathways in CCRC
will help to identify potential new targets. Furthermore, since activation of HIF by physiological hypoxia or other
oncogenic mechanisms is common in many cancers, CCRC provides an important paradigm for studying the role of
HIF in cancer generally.
Planned training and mentoring arrangements
The Oxygen-sensing laboratory has an international reputation in the field of hypoxia signalling, is situated adjacent
to a major national centre for high-throughput sequencing and is in close proximity to the clinical centre of urooncological activity in Oxford. All proposed techniques are currently employed in the laboratory and adequate
instruction will be given. The student will gain experience of modern genome-wide analyses, using state-of the-art
platforms such a microarrays and next-generation high-throughput sequencing as well as a broad range of molecular
and cellular biology techniques. The project would particularly suit a trainee, wishing to translate the output from
these exciting new technologies into a more clinical setting and would complement clinical training in a broad range
of specialities encompassing oncology, urology and renal medicine.
Selective sensitization to ionizing irradiation of cancer cells defective in DNA damage signalling and DNA damage
response pathways
Dr. Grigory Dianov-Scientific Supervisor
Prof. Gillies McKenna-Clinical Supervisor
Summary
We propose that a substantial number of cancers should fall into one of two categories: cancers with non-functional
DNA damage signalling (ARF-pathway deficient) and cancers with a non-functional DNA damage response (p53pathway deficient). Our preliminary data indicate that cancer cells within these two groups may be identified by
specific biomarkers and that they respond differently to DNA damage induced by ionizing radiation. As a
consequence, these cells may be specifically sensitized to ionizing radiation by selective inhibition of proteins
essential for their survival. We propose to further investigate the difference in DNA damage responses, to develop
selective biomarkers and to investigate selective sensitivity of these two groups of cancer cells to specific
combinations of ionizing radiation and DNA repair/DNA signalling inhibitors. The goal of this study is to design
research-based individualized treatment for cancer patients.
Background
Genetic instability, frequently associated with the inability of cells to properly repair DNA damage, is a hallmark of
cancer cells. In normal cells, the accumulation of damaged DNA activates the cellular DNA damage response that
leads to an accumulation of p53 protein and results in cell cycle delay to accommodate for DNA repair, or apoptosis if
the damage is too heavy. To avoid these consequences, cancer cells frequently inactivate either the DNA damage
signalling pathway or the DNA damage response implementing mechanisms. This allows cancer cells to initiate
replication of their DNA without delay, even though the DNA is still damaged. In many cases, this subsequently leads
to collapse of replication forks and the formation of DNA double strand breaks. Therefore, many cancer cells are
known to be sensitive to inhibitors affecting non-homologous end joining (NHEJ) or homologous recombination (HR).
Loss of p53 function is the most frequent event in cancer cells that leads to a knockdown of the DNA damage
response executing mechanism. However, less is known about the mechanisms responsible for DNA damage
detection and signalling. We have recently discovered that ARF/p14 protein, a well-known oncogene frequently
mutated in many cancers, plays a major role in a chain of enzymatic reactions involved in DNA damage signalling.
We discovered that as long as unrepaired DNA strand breaks are detected, this leads to an accumulation of a stable
form of ARF. Since p53 stability is controlled by Mdm2 ubiquitylation and Mdm2 activity is inhibited by ARF, ARF
induction upon unrepaired DNA strand breaks leads to p53 stabilization and activation of the DNA damage response
implementation, including cell cycle delay of DNA replication that is required for completion of DNA repair.
The DNA damage signalling and DNA damage response network is extremely important for the prevention of the
replication of damaged DNA. As a consequence, cancer cells frequently inactivate either ARF-dependent DNA
damage signalling or the p53-dependent execution of the DNA damage response. Inactivation of either ARF or p53
functions in cancer cells leads to two different scenarios of the cellular response to DNA damage. Firstly, nonfunctional ARF will lead to an accumulation of DNA damage but the signal would not be transmitted to p53. In this
case their survival will be highly dependent on DNA double strand break repair and therefore these cells should be
sensitive to a combination of ionizing radiation plus inhibitors of HR and/or NHEJ. These cells should also have low
levels of p53. In the case of cancer cells with non-functional p53 (either deleted or mutated), the DNA damage
signalling pathway will operate normally to induce ARF transcription, but in the absence of p53, ARF levels would not
be down-regulated and will remain high. Two biomarkers should therefore be characteristic for such cells: 1.
Increased expression of ARF; 2. Low levels or mutant p53, and if both are overexpressed at the same time, than p53
should be mutant.
Aims
1. To further investigate the difference in the DNA damage response within two predicted groups of cancer cells.
2. To develop pathway specific biomarkers that will allow discrimination between cancer cells deficient in DNA
damage signalling or the DNA damage response.
3. To investigate selective sensitivity of these two groups of cancer cells to specific combinations of ionizing
radiation and DNA repair/DNA signalling inhibitors.
Research plan
During the first two years we would like to further investigate the mechanism of the DNA damage response in p53and ARF-deficient cells because further work is required to develop our model in full detail. At the same time, we will
also establish a panel of melanoma cancer cell lines and identify and verify biomarkers that will allow us to categorize
DNA signalling and DNA damage response-deficient cell lines. During years two and three, we will test the effect of
ionizing radiation and DNA repair inhibitors on selected cell lines representing cells deficient in DNA damage
signalling or in the DNA damage response.
Expected Impact:
The long term goal of this study would be to develop cheap and simple clinical tests for identifying cancers that falls
within defective DNA signalling or DNA damage response categories, and to initiate clinical trials for improving
cancer treatment using a combination of ionizing radiation and specific clinically approved chemotherapy.
IGF-1R as a mediator of chemoresistance in melanoma
Dr V Macaulay (scientific supervision)
Professor Mark Middleton (clinical supervision)
Abstract: The type 1 IGF receptor (IGF-1R) promotes proliferation and cell survival, and IGF-1R inhibitory drugs are
undergoing clinical evaluation. IGF-1R inhibition is known to enhance chemo-sensitivity; this effect has been
attributed to well-characterized effects on apoptosis induction, but also appears to involve aspects of the cellular
response to DNA damage. This project will characterize the mechanism by which IGF-1R mediates resistance to
temozolomide (TMZ), a methylating agent used in clinical treatment of melanoma. The findings will have
implications for selection of patients for TMZ therapy, and development of approaches to overcome TMZ resistance.
Project proposal: Previous work has shown that depletion or inhibition of type 1 IGF receptor (IGF-1R) influences the
repair of DNA damage, as is induced by cancer treatments including ionizing radiation and many forms of
chemotherapy. This project focuses on damage induced by dacarbazine or its derivative TMZ, methylating agents
that are used clinically, although with limited activity, inducing responses in only 10-15% of melanoma patients. TMZ
generates toxic O6-methyguanine (O6MeG) in DNA that is removed by O6-methylguanine-DNA methyltransferase
(MGMT). Persistent O6MeG triggers mis-pairing and futile cycles of mismatch repair, leading to formation of
replication-associated DNA double-strand breaks (DSBs). Our data indicate that IGF-1R inhibition enhances the
sensitivity of BRAF wild-type and mutant melanoma cells to TMZ, both in vitro and in melanoma xenografts in vivo.
This effect appears to be MGMT-independent, and is associated with delayed resolution of foci formed by RAD51, a
recombinase that plays a key role in the strand invasion step of homologous recombination (HR) repair of DSBs. We
speculate that IGF-1R inhibition influences a post-RAD51 step of HR, or generates recombination intermediates that
are more complex and difficult to resolve. Investigation of these possibilities will involve new collaboration between
the IGF (Macaulay) and DNA Damage and Repair (McHugh) groups. The clinical fellow will take three approaches to
investigate effects of IGF-1R inhibition on the repair of TMZ-induced DSBs:
1) Characterize effects of IGF-1R inhibition on the DNA damage response. Chromatin fractionation and
immunofluorescence assays will be used to investigate the loading of repair proteins onto chromatin, the formation
of foci by HR components including RAD52, RAD54 and RAD51 paralogs, and to determine effects of IGF-1R inhibition
on DNA replication and replication fork damage, in melanoma cells treated with TMZ and other drugs that cause
lesions capable of blocking replication.
2) Define the signalling pathways implicated in mediating effects of IGF-1R on the damage response. Chemical
inhibitors and siRNAs will be used to block individual signalling pathways downstream of IGF-1R in BRAF wild-type
and mutant melanoma cells. Involvement of the recently-identified phenomenon of IGF-1R nuclear translocation will
be tested using IGF-1R-deficient WM35 melanoma cells expressing IGF-1Rs modified to manipulate their subcellular
localization.
3) Perform IGF-1R immunohistochemistry (IHC) on sections of melanoma from patients who showed response or
resistance to clinical treatment with TMZ or its analogue dacarbazine.
Clinical and translational relevance: This project will define the contribution of IGF-1R to TMZ resistance, and will
inform evaluation of IGF-1R inhibitors in treatment of patients with melanoma.
Planned training and mentoring: The clinical fellow will be trained by Macaulay lab members in IGF biology, cell and
molecular biology and IHC, and by Peter McHugh’s group in assays for DNA metabolism and repair. The appointee
will contribute to a weekly outpatient clinic, and will attend the WIMM Methods and Techniques course, and
University courses on statistics and thesis writing. If an Oncologist, the appointee will be mentored by Dr Macaulay,
otherwise by a clinical consultant appropriate to his/her clinical speciality.
Detection, genesis and eradication of melanoma stem cells
Colin Goding (Scientific supervisor) Eric O’Neilll (Scientific co-supervisor)
Mark Middleton (Clinical Co-Supervisor)
Abstract
A major challenge to effective anti-cancer therapy is tumour cell heterogeneity. Within cancers, multiple subpopulations of cancer cells may co-exist, each with differing biological properties. Some may exhibit features of
differentiation, others proliferate, while some may possess stem cell-like properties, able to initiate new tumours
and provide a pool of therapeutically resistant cells. Understanding the origins of cancer cell heterogeneity and how
it can be managed to provide more effective treatments is a key issue.
Although genetic heterogeneity will play a role, recent evidence suggests that the identity of cancer cell
subpopulations is dictated by the tumour microenvironment, and that cells can switch phenotypes. The Goding Lab
has identified key transcription factors as drivers of phenotype-switching in melanoma, one of the most highly
aggressive and increasingly common cancers, enabling the identification by multicolour immunofluorescence of
stem-like cells within melanoma tissue and in primary short-term cultures derived from dissociated melanomas. The
O’Neill Lab has determined that that the Hippo signalling pathway, implicated in control of organ size, is a critical
determinant of ‘stemness’. Preliminary results also suggest that Hippo signalling is a key driver of stemness in
melanoma.
Project proposal
1. Determining the role of Hippo signalling in melanoma sub-population identity.
By using the characteristics of physiological melanocyte stem cells as a guide we have identified a combination of
markers that together enable us to detect stem-like cells in melanoma tissue. Stem-like cells appear to be relatively
rare (less than 0.1-1% of the tumour) and are frequently found close to vessels. On dissociation of melanomas, these
stem-like cells can be identified in short-term cultures and preliminary results also indicate that they may represent
around 0.1% of cells in established cell lines. Significantly the Goding lab has developed lentivirus-based dual colour
fluorescent reporters that distinguish stem cells from non-stem cells in culture. Moreover, preliminary experiments
undertaken with the O’Neill lab have indicated that ectopic expression of one of the key components of the Hippo
signalling pathway in melanoma cells in culture leads to a stem-like phenotype. Using the fluorescent reporters to
isolate stem-like cells by FACS from dissociated primary melanoma tissue provided by Mark Middleton, we will use
RNA-seq to determine the repertoire of genes that characterise this stem-like population or whether stem-like cells
isolated from different patients are similar. In a parallel approach we will use an inducible expression system to
activate Hippo signalling in melanoma cells in culture and compare the gene expression profile of the resulting stemlike cells to that present in stem-cells isolated directly from patients. Bioinformatics analysis, undertaken by a
dedicated Ludwig bioinformatician in collaboration with the CRF, will be used to determine the similarity/differences
between the Hippo-induced melanoma stem-like cells and natural melanoma stem-like cells isolated directly from
tumour tissue.
In addition the Goding lab has determined that around 30% of cells present in a melanoblast cell line exhibit stemlike properties based on the dual-colour reporter system. We will use a combination of siRNA or shRNA-mediated
depletion of Hippo pathway components to activate/inactivate Hippo signalling in these cells and determine how this
affects their differentiation/stem-like status and their proliferative and invasive properties. The outputs will also be
monitored using antibodies against key melanoma transcription factors that define melanoma sub-population
identity as well as hippo pathway components, and key hippo-regulated signalling molecules (eg beta-catenin) that
play a crucial role in determining melanoma proliferation and sub-population identity.
2. Microenvironmental signals regulating Hippo signalling in melanoma
Our current model predicts that activation of hippo signalling generates melanoma stem-like cells that are potentially
highly invasive and should have the capacity to initiate tumour formation in xenografts and generate heterogenous
tumours. However what microenvironmental signals activate hippo signalling is unknown. Using hippo pathway
reporters generated by the O’Neill lab we will screen for effects of candidate microenvironmental signals including
hypoxia, nutrient deprivation, cytokines and other signals generated by infiltrating cells from the immune system.
Since the Goding lab can identify melanoma stem cells in frozen melanoma tissue provided by Middleton, similar
samples will be probed using antibodies generated by the O’Neill lab against key components of the hippo signalling
pathway. The CRF will therefore collaborate with others in the Goding and O’Neill lab to determine whether
activation of the pathway, as indicated by the switch from nuclear to cytoplasmic subcellular localisation of the hippo
pathway YAP or TAZ transcription cofactors, occurs in stem cells or other melanoma sub-populations in melanoma
tissue samples.
The frequency and distribution of stem-like cells and hippo signalling-positive cells will be evaluated in fresh frozen
(n=50, all annotated) and paraffin embedded (n>1500, all annotated) tumour samples held in the melanoma
biobank.
3. Medium throughput screens aimed at targeting stem cells and hippo pathway signalling
With the completion of the TDI in 2013 substantial opportunities will exist for screening for drugs that exert specific
biological effects. We have already successfully undertaken a low throughput 350 kinase inhibitor screen for
compounds that affect MITF levels or sub-cellular localisation based on automated microscopy and
immunofluorescence. We therefore aim to extend this screen to a 5000 compound library comprising FDA approved
drugs, the rationale being that many drugs may have effects in biological assays that have not previously been
tested. For example some drugs may completely eradicate specific subpopulations of melanoma cells, but leave
other subpopulations intact to go on to regenerate tumours. The efficacy of such drugs against specific
subpopulations would not therefore be recognised in clinical trials. Since we can assay for the presence of melanoma
stem-like cells, even within established cell lines, we can screen for drugs that eradicate the low proportion of stem
like cells, or that inhibit or enhance hippo pathway signalling which would be expected to affect the proportion of
stem like cells in a population. Since the Goding lab has developed a red/green reporter assay that marks the
proportion of stem cells in culture a simple screen can be performed rapidly, for example using the melanoblast cell
line that contains 30% of cells in a stem-like state. A hit would be defined as a drug that significantly switches the
ratio of red to green cells. Any drugs identified in a first round screen will be validated on dissociated primary
melanoma material using immunofluorescence to identify the proportion of stem like cells, and will be tested for
their effects on hippo signalling using hippo pathway reporter assays developed by the O’Neill lab. Validated hits will
be evaluated in patients amenable to biopsy or due to undergo surgery for their melanoma, to evaluate the impact
on stem like cells (see below).
Clinical/ Translational relevance of project and new collaboration
The aim of this project is to determine first the role of the Hippo signalling pathway in melanoma sub-population
identity and second to screen for drugs that may drive elimination of resistant cells within tumours. The project is
therefore of direct clinical interest. First, the analysis of the stem cell transcriptome should enable the identification
of cell surface and other markers that may facilitate the detection of this potentially therapeutically resistant
population of cells in tumour samples; and second, since the compound library to be screened contains 5000 FDA
approved drugs any positives arising from the screen may be rapidly incorporated into clinical trials. The project
therefore aims to build on the basic science undertaken by the Goding and O’Neill labs and rapidly translate the
findings to clinical use.
Lead compounds, since these are already formulated, will be assessed for their effect in tumours within the existing
phase 0 melanoma trial framework, and developed as combinations with standard therapeutics for melanoma.
Planned training and mentoring arrangements
Primary supervision will be provided by Prof. Goding and Dr O’Neill who will provide the general strategy for the
project, and jointly meet with the CRF at least weekly to discuss progress and trouble shoot. Day to day training in
the technical aspects of the project will be provided by Dr Rasmus Freter who has succeeded in identifying
melanoma stem-like cells in tumours and in culture and who has extensive experience in the techniques to be used.
The Goding and O’Neill labs between them contain many post-docs and students who are experts in melanoma or
hippo signalling and who will also provide additional technical and intellectual input/support when needed.
Mark Middleton will provide clinical supervision and oversee clinical trial development. He will meet fortnightly with
the CRF as well as Goding and O’Neill to assist in the clinical and translational aspects of the project.
In addition to training in the scientific aspects of the project the Goding lab also provides all students and post-docs
with training in complementary skills, including: oral presentations at weekly lab meetings, the Ludwig Institute
weekly seminar program and monthly journal club, paper and grant writing, and data management and presentation,
refereeing etc. Middleton’s group will also provide training in clinical trial conduct to GCP, trial design, protocol
development, regulatory submission, data management and reporting and the incorporation of translational endpoints into clinical studies.
The role of changes in the mitochondrial genome in tumour responses to local microenvironments and treatment
Professor David Vaux (Scientific Supervisor)
Professor Adrian Harris (Clinical Supervisor)
Abstract – the mitochondrial changes that underpin the increased dependence of cancer cells on aerobic glycolysis
for energy generation remain uncertain. Subtle effects of accumulated mutations in the small mitochondrial
genome, known to encode components of the critical oxidative phosphorylation pathway for ATP production, may
support this shift; strong selection pressures due to physiological (hypoxia) or therapeutic (radio-, chemo- or
antiangiogenic- therapy) stress may promote either cell adaptation and survival due to decreased mitochondrial
activity, or apoptotic death. To understand how the balance between these outcomes is established, and ultimately
how it might be manipulated, we will use next-gen sequencing of the mitochondrial genome at high fold-coverage to
examine emergence and propagation of patterns of mtDNA mutations in experimental models of cancer therapy and
in patient tumour samples.
Project proposal – Our observation of an active pool of the DNA repair-orchestrating protein BRCA1 co-localised with
nucleoids of mitochondrial DNA (mtDNA) in the mitochondrial matrix in normal human cells (Coene et al (2005) Mol
Biol Cell 16 997-1010) prompted us to study mtDNA in breast cancer cells. A multiplexed, ratiometric TaqMan qRTPCR assay (based on Mambo et al (2003) PNAS 100 1838-43) showed increased mtDNA damage in cells lacking
functional BRCA1, together with reduced repair after genotoxic challenges. A recent CRC Development Fund award
has enabled us to confirm and extend this observation by MiSeq sequencing of the entire mtDNA genome at >4,000
fold coverage in samples from human breast cancer cells expressing no BRCA1, ubiquitin-ligase dead point mutant
BRCA1 (I26A mutant) or functional wild type BRCA1. Initial analysis of mtDNA sequences from these cells, with and
without a genotoxic challenge and a recovery period, confirmed that we could detect sites with an increased
probability of mutation, presumably caused by inaccurate repair after DNA damage. The resulting low level
heteroplasmy could be readily detected at a frequency of <0.2% and showed a strongly non-random distribution as
well as site-specific vulnerability to different DNA damaging agents (with 10-fold differences between treatment with
the experimental agents alloxan or 4-nitro-quinoline-1-oxide, for example). These results establish a significant role
for BRCA1 in protection of the mitochondrial genome, but more generally offer a tool to analyse human mtDNA
vulnerability to a wide range of stresses, either alone or in combination.
The experimental approach in this project fosters a new collaboration between preclinical and clinical research
groups and falls naturally into two parts; in one we will use in vitro and in vivo model systems of human cancer
development and growth that can be manipulated in mimicry of human anti-cancer therapy. In the other we will
examine the clinical consequences of genetic selection by micro-environment operating on these increased mutation
rates to generate mtDNA mutations in vivo, by analysing primary and metastatic tumours resected from patients.
We will compare model tumour cell growth in vitro in cell culture (both monolayer culture and the more
physiological spheroid 3D culture) and in vivo using MDA231 [triple receptor negative] and HCC-1937 [BRCA- mutant,
see below] xenografts in nude mice. MiSeq sequencing of the entire mitochondrial genome will be used to follow
the emergence of mtDNA mutations, which will be analysed in time-series and in cross-section to detect shared
patterns of co-selection. By comparing untreated controls with samples treated with clinically-relevant DNAdamaging agents (including cisplatinum, cyclophosphamide, temozolomide, anthracyclines, and radiation), antiangiogenic (anti-VEGF) therapy leading to hypoxic stress, or drugs such as DCA that block mitochondrial function, we
will test for the emergence of reproducible patterns of mutation. The functional consequence of these emerging
changes will be followed in the culture models by cell cycle and apoptosis analysis (flow cytometry), mitochondrial
function analysis (live cell confocal fluorescence microscopy and ImageStream using dyes sensitive to mitochondrial
trans-membrane potential and reactive oxygen species) and metabolic profiling (glucose/lactate ratio to detect any
shift towards anaerobic glycolysis). To separate effects of nuclear and mitochondrial mutations we will re-analyse
cells in which mutation-bearing mitochondria are delivered to an untreated control nuclear background using the
cybrid mitochondrial transfer technique (Ishikawa et al (2012) J Bioenerg Biomembr DOI 10.1007/s10863-012-94686), which has already been used to confirm the role of an mtDNA mutation in regulating metastasis (Ishikawa et al
(2008) Science 320 661-).
To delineate the role of BRCA1 in these events we will compare a human breast cancer cell line (HCC-1937) that
naturally lacks functional BRCA1 with our existing derivative cell lines engineered to express either functional or
enzymatically inactive BRCA1. Once again, we will distinguish nuclear from mitochondrial vulnerability using the
cybrid transfer method.
To translate and extend these results into the clinical arena we will sequence the entire mitochondrial genome from
existing paired samples of primary breast cancer and lymph node metastases from a carefully selected group of
thirty cases chosen to allow testing of different types of breast cancer, in which there may be a range of both
mutation risk and mutation sites. This analysis will include estrogen receptor (ER) –ve versus ER +ve tumours and
triple receptor negative tumours, an important poor prognosis group sharing some characteristics with BRCA1 -/tumours. This approach will enable us to establish the scale and pattern of mtDNA mutation that develops in natural
tumours under the selection pressure of the micro-environment at the primary site and in metastases that grow in
draining lymph nodes. Differences in the distribution and frequency of the mtDNA mutations detected between
these sample groups and the experimental xenograft tumours will identify the effects of in vivo selection pressures,
whether due to intrinsic local environmental factors or as a result of therapeutic intervention.
Clinical/Translational relevance of project – The role of metabolism in cancer growth, and particularly the switch to
aerobic glycolysis, has become increasingly recognised, both in the biology of cancer and as a potential therapeutic
target. This switch is accompanied by reduced mitochondrial metabolism, but nevertheless some residual
mitochondrial function is necessary for optimal tumour cell growth. Although there is a reduction in oxygen
consumption and electron transport chain function, the Krebs cycle is still necessary for fatty acid oxidation for a
considerable contribution to ATP generation, even in hypoxic circumstances. Correlating mtDNA changes and effects
on the electron transport chain will provide a critical basis for understanding the biology of metabolic changes in
hypoxia. Finding such changes in primary tumours, especially if there are further changes in secondary deposits due
to micro-environment differences, will provide important insights into potential therapeutic approaches. For
example, selective interference with mitochondrial function has been a difficult challenge but if there are
reproducible mutational differences between tumour and normal mitochondria, this provides new therapeutic
opportunities. Moreover, if mtDNA mutations underpin the heterogeneity and variability in stress-induced free
radical generation, screening for them would help to identify tumours that might be responsive to inhibition of
glycolysis pathways, for which new drugs are currently in development.
Samples available from our clinical studies have detailed gene expression data that can be related to the
mitochondrial genes that are nuclear-encoded and to metabolic genes involved in glycolysis, fatty acid metabolism
and pentose shunt. This will allow assessment of the relationship of the mitochondrial mutations to other key
pathways that may be co-regulated. These may provide a novel target for intervention in metabolism and selective
toxicity. They will help classify patients into those most likely to be responsive to inhibitors of glucose metabolism.
Although we will be specifically studying mutations induced by clinically used drugs, it is important to point out that
the genome is mutated before drug exposure and that this may be a new way of classifying tumours with key genetic
changes to their metabolism.
Planned training and mentoring arrangements - the Clinical Research Training Fellow will join a basic science lab
with a strong mentoring ethos, as has the WIMM lab, including supervision and training of >60 FHS research project
students, as well as part II students, visiting academics and a small number of tightly integrated graduate students
(currently three in the group, 100% 4-year submission rate). Mentoring arrangements include daily contact with
senior postdocs, weekly lab meetings alternating with Prof Harris, 12 weekly joint lab presentations, annual
presentations at the graduate symposium and DPhil Day, as well as regular meetings with a departmental advisor
and a College mentor. The Fellow will be actively encouraged to use the full range of graduate skills training courses,
including professional skills training off-site (e.g. Grad School), together with focused training in experimental
methods (Departmental training courses, Techniques Days, etc) and software for data analysis (CBRG maintains its
only South Parks Road office within the Dunn School; the Fellow will also have access to the huge range of OUCS
courses).
Investigating the role of ASPP/p53 and Dll4/Notch cross talk in patients response to anti-VEGFR based therapy
Professor Xin Lu and Dr. Sarah De Val (Scientific Supervisors) Ludwig Institute for Cancer Research
Professor Adrian Harris (Clinical Supervisor)
Abstract
The tumour suppressor p53 and the cell fate determination Notch pathway play key roles in cancer metastasis. As
transcription factors, both p53 and activated Notch regulate the expression of a large array of target genes. In
response to stress signals, activation of p53 regulated genes induces various cellular responses that are involved in
regulating biological processes such as metabolism, cellular senescence and apoptosis. The activation of Notch
signalling by its ligand such as Dll4 induces endothelial cell proliferation and tumour angiogenesis. Since cancer
therapy induces stress and also alters Notch signalling, the cross talk between p53 and Notch pathway is critical in
dictating tumour response to therapy.The functional interplay between p53 and Notch is likely to be important in
determining patients’ response to anti-angiogenesis based therapy. The therapeutic potential of inhibiting the
activities of vascular endothelial growth factor receptor (VEGFR) pathway led to the development of Bevacizumab
(an anti-VEGF antibody). Unfortunately, Bevacizumab resistance does occur and the underlying mechanism of
resistance remains largely unknown.
Our previous studies showed that signalling through Dll4 induces resistance to Bevacizumab in preclinical models.
We have previously shown that Dll4 is regulated by hypoxia, VEGF and FGF and markedly upregulated in human
tumour vasculature. There is cross talk between Dll4 and VEGF, with VEGF up-regulation of Dll4 and Dll4 downregulating VEGF receptor (VEGFR). Dll4 signalling also stabilises vessels, reduces proliferation and reduces sprouting.
Interestingly, VEGFR and Notch have been recently identified as novel p53 target genes. Mutation in the DNA binding
domain of p53 often inactivates or alters its transcriptional activity. The transcriptional target selectivity of p53 is
also regulated by the evolutionarily conserved ASPP family of proteins that bind the DNA binding domain of p53.
These data suggest the existence of cross talk between p53 and Notch pathways in controlling tumour metastasis
through their ability to modulate tumour angiogenesis. Transcriptional activities of p53 and Notch are likely to play a
key role in determining tumour’s response to anti-VEGFR based therapy. We therefore hypothesize that the
mutation status of p53, the expression levels of the ASPP family of proteins and Notch ligands, including Dll4, are
likely to be important molecular signatures that can predict tumours’ response to anti-VEGF based therapy. To test
this hypothesis, we will take the advantages of the expertise among our three groups, our established preclinical
experimental models and the clinical samples and information generated over the years. In particular we will focus
on the following three aims.
Aim 1: Use the transgenic mouse models that either contains tumour derived p53 mutation or defective in
expressing ASPP proteins to examining the impact of ASPP/p53 pathway in regulating Notch mediated angiogenesis
in vivo.
Aim 2: Use well established human endothelial cells and the standard mouse aortic assay to manipulate the activities
of p53 and Notch using RNAi or genetic deletions to investigate the cross talk between ASPP/p53 and Dll4/Notch
pathways in vitro.
Aim 3: Examine the mutation status of p53, the expression levels of ASPP and Dll4 and their association to patients’
responses to anti-VEGF based therapy.
Plan of investigations.
Investigating the role of ASPP and p53 mutation in controlling angiogenesis in vivo
Although p53 is known to regulate the expression of VEGFR and Notch, its role in angiogenesis remains unclear as
p53 null mice develop normally. Interestingly, there is emerging evidence to suggest that cells expressing mutant p53
function differently from p53 null cells, supporting the notion that mutations in p53 differs from p53 deletion.
Around 50% of human tumours have p53 mutation other than deletion, and the majority of p53 mutations occur in
metastatic tumour cells. It is therefore important to investigate whether mutant p53 is involved in controlling
angiogenesis. The ASPP family of proteins bind the DNA binding domain of p53, a region that harbours around 80%
of tumour derived mutations, and alter the target selectively of p53. ASPP2 binds Par3 and controls the integrity of
apical polarity and tight/adherence junctions in vitro and in vivo.The ASPP family of proteins could also play a role in
controlling both p53 dependent or independent angiogenesis. To test this hypothesis we will take the advantage of
an established retina angiogenesis mouse model in Dr. Sarah De Val’s group using the following transgenic mice that
are available in Prof Xin Lu’s group; p53(+/+), p53(+/-), p53(-/-), p53His172/p53wt, p53His172/p53His172,
ASPP2(+/+), ASPP2(+/-) and ASPP2(-/-). This study will demonstrate the potential role of p53 mutation status and
ASPP2 expression level in controlling angiogenesis. Since we have already obtained preliminary data showing blood
vessel leakage in ASPP2 mutant mouse brain in vivo, it is likely that ASPP2 status will affect angiogenesis in vivo.
Investigating the cross talk between p53 and Notch in vessel formation in vitro
It has been well established by Prof Harris’ group that Dll4/Notch stabilises vessels, reduces proliferation and reduces
sprouting. Prof Harris’ group also established human umbilical endothelial cell (HUVEC) cultures and aortic assays to
study the mechanistic insight into angiogenesis process in vivo. We will therefore use these in vitro systems to study
whether the cross talk between p53/ASPP and Dll4/Notch will have a large impact in angiogenesis. Using RNAi or
expression plasmids of p53, ASPP, Dll4 and Notch to either reduce or increase the expression levels of these proteins
in HUVECs will allow us to address whether the proteins or associated pathways will affect the branching or
sprouting properties of HUVECs. The expression levels of ASPP2, p53 or mutant p53 on angiogenesis can also be
tested in mouse aorta derived from the transgenic mice (see above) in a standard aortic assay that has also been
established in Prof Harris’ lab. By incubating HUVECs or aorta with Dll4 in the presence or absence of ASPP2, p53 or
mutant p53, we will investigate the cross talk between p53 and Notch in regulating angiogenesis in vitro. This study
will illustrate whether and to what extent ASPP/p53 and Dll4/Notch interact to regulate angiogenesis.
Investigate the potential role of ASPP/p53 and Dll4/Notch as molecular signatures of tumour response to anti-VEGF
based therapy
Over the years, Prof Harris’ group have generated tissue microarrays of the major tumour types including non-small
cell lung cancer, colon cancer, breast cancer and prostate cancer with long term follow up and resistance to antiVEGF therapy. Notch signalling status has also been examined in a number of tumour cohorts, predominantly by
scoring Dll4 staining of blood vessels and determining tumour vessel density via CD31. We plan to use the same
cohorts of tumour arrays with known Notch signalling status to study the link between ASPP and p53 expression
levels, using anti- ASPP and p53 antibodies generated and characterised in Prof Lu’s group. Since p53 and Notch can
only perform their transcriptional activity when located in the nucleus, whereas ASPP2 protein shuttles between
cell/cell junction, cytoplasm and nucleus, we will pay special attention to cellular localisation of ASPP, p53, Dll4 and
Notch. The association between Dll4 expression level, nuclear accumulation of Notch and nuclear p53 and ASPP2 will
be examined both in tumour cells and tumour blood vessels. This study will enable us to establish whether the
expression levels and/or locations of ASPP and p53 are linked to Dll4/Notch activity. Together, the expression
patterns of the above mentioned molecules and patients survival and response to anti-VEGF treatment (available
from an in house breast cancer study and also TMA based on 80 renal cancer patients treated with sunitinib) will
provide clues to whether ASPP/p53 and Dll4/Notch are molecular signatures that may allow us to stratify patients
or/and to predict patients’ response to anti-VEGF based therapy.
Clinical/ Translational relevance of project
The therapeutic potential of inhibiting the activities of vascular endothelial growth factor receptor (VEGFR) pathway
led to the development of Bevacizumab (an anti-VEGF antibody). Unfortunately, Bevacizumab resistance does occur
and the underlying mechanism of resistance remains largely unknown. We hypothesize that the mutation status of
p53, the expression levels of the ASPP family of proteins and Notch ligands, including Dll4, are likely to be important
molecular signatures that can predict tumours’ response to anti-VEGF based therapy. Selecting patients who are
most likely to benefit from anti-VEGF therapy is a step towards personalised cacner treatment. New insights into the
mechanism of resistance to Bevacizumab may further identify additional targets for novel drug development.
Planned training and Mentoring arrangements
This proposed OCRC Clinical Research Fellowship forms the basis for a new collaboration between the Ludwig
Institute for Cancer Research (LICR) and Prof Harris’ laboratory. Prof Harris has published extensively on Notch
signalling mediated angiogenesis in cancer. Prof Lu’s group is one of the world’s leading p53 research groups and
discovered the ASPP family proteins. Dr De Val’s group investigates transcriptional networks governing vascular
growth during development. Basic science supervision will be provided by ProfLu, Prof Harris and Dr de Val while
clinical mentoring will be undertaken by Professor Harris. Training in the well-established techniques described in
this project summary will be arranged by the supervisors in collaboration with experienced post-doctoral researchers
and DPhil students.
Risk factors for ovarian cancer by histological phenotype in the Million Women Study
Professor Dame Valerie Beral (Scientific Supervisor)
Dr Ahmed Ahmed (Clinical Supervisor)
Abstract
This project is to investigate how a range of different factors affect a woman’s risk of developing ovarian cancer. The
primary aim of the project is to examine the association between various lifestyle factors and incident ovarian
cancers among 1.3 million middle-aged women recruited into the Million Women Study, and to investigate whether
these associations vary depending on the histological phenotype of the tumour. A secondary aim is to explore
genetic risk factors for ovarian cancer, within a subset of the cohort.
Project proposal
This project will examine data on incident cases of ovarian cancer in a large prospective cohort, the Million Women
Study (MWS). Study participants were recruited, on average, in 1998 when they were 56 years old and have now
been followed for ten years for incident cancer. All participants completed a comprehensive initial questionnaire
regarding socio-demographic and lifestyle factors. Three follow-up questionnaires have also been sent to
participants, about 3 years apart. Through NHS Central Registries, cancer registrations and deaths have been
recorded.
This project will involve examination of factors associated with incident ovarian cancer in a large cohort of (mostly)
postmenopausal women, with particular emphasis on variation by histological phenotype. For some exposures, this
will represent the first time that these associations have been explored with this dataset. For other exposures (e.g.
oral contraceptive pill and hormone replacement therapy), there has been previous work using data from MWS, but
new analyses are needed to take account of several years’ worth of further follow-up data.
In addition, blood samples have been obtained for a subset of the cohort, and material stored for genetic analysis.
There is thus the potential for exploration of associations between specific genetic polymorphisms and incident
ovarian cancer.
Clinical/ Translational relevance of project
The histopathological classification of ovarian cancer is currently contentious, with several novel theories regarding
the pathogenesis of different phenotypes (including possible extra-ovarian origins). Thus far, the evidence base for
these new theories has tended to be limited to histopathological case series and molecular studies with relatively
small sample sizes. The Million Women Study, with over 5000 cases of incident ovarian cancer, allows for the
exploration of risk factors split by histological phenotype to an extent not feasible in any other study. Greater
understanding of variation in risk factors by histological phenotype may help to elucidate underlying molecular
differences. This, in turn, could facilitate targeted therapies, and potentially earlier detection and/or prevention.
Planned training and mentoring arrangements
The main emphasis of research in the Cancer Epidemiology Unit is on providing large-scale reliable evidence on the
relationship between common exposures, such as reproductive factors, diet and the use of oral contraceptives and
hormone replacement therapy, and common conditions of public health importance such as cancers, cardiovascular
disease and fractures. The successful applicant will receive training in epidemiology, including literature reviews,
academic writing, and statistical methods for data analysis and will work with a strong interdisciplinary team of
researchers with expertise in epidemiology, statistics, clinical medicine, biochemistry and genetics. Overall
supervision will be provided by Prof Beral (Director), with additional mentoring by other members of the Unit.
Molecular selection of therapy in colorectal cancer: Bedside to bench and back again
Professor Tim Maughan (Clinical Supervisor)
Professor Walter Bodmer (Scientific Supervisor)
Abstract
Aim: to develop effective combination therapies for patients with molecular subtypes of colorectal cancer based on
molecular characterisation of retrospective and prospective clinical samples and hypothesis driven cell line testing in
the laboratory for application into clinical trials in early and advanced disease. The fellow will participate in the
management and treatment aspects of the MRC FOCUS4 trial of molecularly selected therapy in advanced colorectal
cancer. In addition, the laboratory project will be discovering the clinical correlates of in depth molecular
characterisation of a cohort of retrospective patients from MRC trials. Primary cultures will be established from
patients on trial and will be characterised to assess how they differ from direct analyses from biopsies and those in
long term culture. Combination therapy sensitivity will be assessed using high throughput screening techniques and
specific biological hypotheses pursued to identify novel combination strategies to treat defined molecular subpopulations within the spectrum of colorectal cancer.
Project Proposal
This CRF would be an integral part of the Maughan Bodmer CR-UK programme and link with the MRC FOCUS4 trial.
The primary aim of the lab programme is to obtain in vitro sensitivity data of novel drug and drug radiation
combination treatments from well-characterized cell lines and to link the cell line genomic, molecular and biological
data to a large cohort of well-characterized clinical samples in order to provide new insights into the fundamental
biology of CRC and be a basis for biomarker driven novel treatment combinations for clinical trial. The FOCUS4 trial
will commence recruitment early in 2013 and is a trials programme designed as a framework for efficient phase II/III
testing for clinical benefit of novel targeted agents in biomarker selected cohorts of colorectal cancer patients. This
framework has been developed in such a way as to be adaptive to the rapidly evolving molecular characterisation or
stratification of cancers into subtypes predictive of response. The integration of the clinical trial with the laboratory
programme provides an exceptional translational environment and will be a unique training opportunity for both
clinical and laboratory aspects of personalised medicine.
The research fellow would have clinical and laboratory commitments. Clinical responsibility (2 days/wk) would
include seeing patients in clinic on FOCUS4, to manage
toxicity and support the clinical evaluation of patients in
order to
gain hands on clinical experience of novel combination
therapy treatments in patients with CRC and facilitating
the obtaining of biopsy samples from these patients on
randomisation and on progression for primary culture
and tumour characterisation. In addition the fellow
would interact with the MRC CTU where there will be a
dedicated trials fellow to do the CTU related data and
design work. The CR-UK fellow should understand the
design issues in the trial and be in a position to take up
one or more clinical aspects of the trial to write up.
In the laboratory (3 days/wk), the fellow will be involved in the following sections of the programme:
1. Molecular characterisation and clinical correlation of colorectal cancer. We are in the process of cutting
sections from 200+ patients from FOCUS 3 which will be analysed in Oxford on the Ion Torrent platform, with full
clinical data from the randomised trial. In addition, we have requested funding for in depth analysis of 450 patients
from COIN (450 gene mutation sequencing, genome wide gene expression and methylation status) in whom we have
full clinical data. The cell lines are about to be analysed (free) at the Sanger Institute for WES, SNP 6 array for CNV,
gene expression and methylation status. The fellow will assist in the bioinformatic analysis of the retrospective
clinical sample results and its association with clinical characteristics and outcome, working with the Department of
Oncology bioinformatics core and with access to the full MRC trials data. These will be among the first clinical trial
analyses to include such extensive genetic testing. We will assess whether the more extensive genetic
characterisation shows good or poor prognostic subtypes, responsive or resistance subtypes (to 5FU, oxaliplatin,
irinotecan, cetuximab and bevacizumab, all of which were used in a randomised strategy in FOCUS3) and assess the
incidence of ‘actionable ‘ findings which could identify clinically
accessible alternate therapeutic strategies.
2. Primary cultures. The technique for establishing primary
culture is already established in the Bodmer laboratory with a
high success rate from small samples from primary tumours.
The next step is to establish the techniques from metastatic
biopsies. The fellow will collaborate with existing staff to
establish primary cultures in the Bodmer laboratory from the
samples taken from the trial patients (see 2) and will collaborate
with ongoing work to characterise the biopsy and early cultured
material and compare with the established cell lines to identify changes in gene expression, methylation and
mutation in early passage.
3. Cell line sensitivity screening. The tumour characterisation and associated functional studies using the in vitro
cell line models will provide new insights into the fundamental biology of CRC and will provide the basis for novel
treatment and tumour property combination predictions for further evaluation in the clinic. We will be undertaking a
range of high throughput, though hypothesis based, screens in the target discovery institute to identify novel
combination therapies. By testing these in the range of the characterised cell lines, we will discover the biomarkers
associated with responsiveness. The fellow will develop and test one or more hypotheses for effective combination
therapy in cell line screening experiments, using the libraries of reagents to be obtained from pharmaceutical
companies, the SGC and other resources. If successful these could form the basis of a new combination therapy
study for early or advanced colorectal cancer.
Clinical/ Translational relevance of project
This personalized medicine approach will identify potential biomarkers that will distinguish responders to specific
therapies for further study and will improve outcomes and reduced toxicity in biomarker driven drug, and
radiotherapy combination trials in colorectal cancer. This will have immediate application in the adaptation of the
FOCUS 4 and other biomarker driven clinical trials in patients with metastatic and adjuvant colorectal cancer.
Planned training and mentoring arrangements
The fellow will be mentored primarily by Prof Maughan who is CI of the FOCUS4 trial programme. The laboratory
work will be overseen in the Bodmer lab where senior post doc Jenny Wilding will ensure the quality of the
laboratory work on a day to day basis and overseen by Sir Walter. In addition the fellow will benefit from the
engagement with the FOCUS4 trial team and the MRC CTU providing an unparalleled opportunity to experience and
understand the design issues about biomarker driven trials.
Modulating tumour immunity by local production of immune mediators from an ‘armed’ oncolytic virus
Prof Leonard W. Seymour (academic supervisor)
Dr Rachel S. Midgley (clinical supervisor)
Abstract
This project will be right at the heart of a translational programme developing a group B oncolytic adenovirus,
thought to kill cancer cells by a process of necrosis. The appointee will gain a detailed understanding of virus activity
in early phase clinical trials and will engineer improved virus variants in the laboratory. Within this project there is a
high expectation that the appointee will take a new and improved virus back into the clinics based on the experience
gained at the interface between laboratory and patient. Training and experience will be gained in all aspects of this
new medical approach including diverse laboratory technologies, clinical trial design, regulatory and ethical
requirements, working with industry and creation of high profile publications.
Project proposal
Background: Oncolytic viruses replicate selectively in cancer cells and lyse them before spreading to infect adjacent
cells and repeat the process. This amplification within the tumour gives a highly favourable biodistribution and
combines powerful cytotoxicity with cancer-selectivity defined by tumour-associated changes. The first group B
oncolytic adenovirus to enter clinical trials, ColoAd1, was administered to the first patient in an intravenous (i.v.)
dose escalation study in September 2012 and 17 additional patients are expected to enter the study within the next
18 months.
ColoAd1 kills tumour cells by a mechanism that is independent of apoptosis and involves early membrane lysis
reminiscent of necrosis, not only providing the possibility of activity in drug resistant cancer but also the opportunity
to break the cycle of sterile inflammation to induce an effective anti-cancer immune response. This is a special
interest of Dr Midgley and Prof Seymour.
Overview of the project: The project will involve a series of trials backed up by focussed translational laboratory
research. The ongoing IV study will be followed by a more mechanistic-based phase zero study involving injection of
a fixed dose of ColoAd1 into primary colorectal tumours 1-2 weeks prior to biopsy or resection, allowing us to
monitor virus infection, spread, selectivity, mechanism of cancer cell killing and early effects on tumour immune
infiltrate. Evidence of both local and systemic immune response will be sought, and regulatory T cells, tissue
associated macrophages and NK cells are of particular interest. This trial has been approved by the Dragon’s den and
can commence within the next 9-12 months. We hypothesise that virus produced through replication within the
tumour will drain via lymphatics and permit infection of tumour micro-metastases in draining lymph nodes,
preventing them from further development and spread.
The appointee will learn techniques allowing him or her to characterise clinical pharmacodynamics of the virus and
to correlate them with the virus biology observed in vitro. In addition he/she will identify factors which may be
limiting successful ‘virotherapy’, with a view to optimising the creation of the next generation virus. As ColoAd1 has
the capacity to accommodate 3.5kb of additional DNA, the appointee will design and engineer modified ‘armed’
versions of the virus, encoding therapeutic proteins at the DNA level so that they are produced only when the virus
enters its replicative cycle i.e. within tumour cells. This will maximise local activity within tumour nodules and also
minimise off-target toxicities. These novel viruses will then be taken through into early phase trials.
Hypothesis: We hypothesise that arming ColoAd1 to produce carefully chosen therapeutic proteins only within
tumour nodules will permit synergistic anticancer activity, potentially increasing the lytic response but also allowing
stimulation of an anticancer immune response.
Experimental plan:
Year 1: Early studies will elucidate the mechanism of cell killing by ColoAd1 in vitro, and will explore whether a similar
mechanism occurs in patients in the colorectal clinical trial. You will also characterise virus activity in patients to
identify any limiting factors.
Year 2/3: We have already produced an ‘engineerable’ version of ColoAd1, so that further genetic modifications to
encode therapeutic proteins are straightforward. The proteins we are considering encoding include cytokines,
interferons and antibodies such as anti-CTLA4 or anti-PD1. We believe that local production of these agents will
elevate the virus’s therapeutic index through direct and indirect immunological effects, thereby enabling synergistic
anticancer activity. Appropriately armed viruses will be characterised in cells in vitro, including in primary human
cells and in human tumour ‘slice’ culture, where freshly resected human tumours are maintained for up to 5 days ex
vivo. This allows study of virus activity in a pre-clinical environment that is very relevant to the clinical setting.
Year 3: ColoAd1 is being developed by the spin out company PsiOxus Therapeutics Ltd, and we will work closely with
the company to raise support for early phase trials of promising novel ‘armed’ variants of the virus. We anticipate at
least one variant will enter phase I study during the 3rd year of this project. The appointee will play a pivotal role in
that study, at both clinical and scientific levels.
Clinical/ Translational relevance of project
This project will operate in a highly translational environment, applying basic cell biology and virological techniques
in the laboratory whilst also setting up early phase clinical trials. Oncolytic viruses provide the ideal tool to apply
genetic technologies for cancer therapy, and the ongoing clinical trials programme of ColoAd1 provides a unique
opportunity to explore challenges in the clinical study and to employ scientific and genetic skills to address them.
Success in this project may translate quickly into improved cancer medications. The appointee will be able to hone
their clinical research skills alongside developing basic science and translational assays in the laboratory and thus the
project is truly translational, working from the bench to the bedside and back again.
Planned training and mentoring arrangements
The appointee will be co-supervised by Prof Len Seymour (scientific) and Dr Rachel Midgley (clinical).
Laboratory training: The appointee will join an active and highly motivated research group with diverse skills in
virology and genetics. He/she will be assigned a postdoc supervisor for laboratory work and will meet with Prof
Seymour regularly, maturing into weekly meetings including monthly meetings jointly with Dr Midgley. The
appointee will also contribute to an active programme of weekly group meetings (each member contributes a
presentation three times a year) and will make annual presentations to larger departmental lecture programmes.
Lab techniques will include: mammalian cell culture, flow cytometry, immunohistochemistry, handling category 2
micro-organisms, cloning, western blotting and quantitative PCR as well as T cell assays and cytokine ELISA.
Bridging research areas and forging new collaborations: This project is truly translational and will make a strong link
between the laboratory and the clinics, working alongside ECMC and BRC personnel in developing new assays and
using clinical results to feed scientific innovation towards the next generation of oncolytic viruses. The appointee will
also be actively involved in collaborations with other immunologists in Oxford, particularly with Enzo Cerundolo and
also including Marc Feldmann and others in the relocating Kennedy Institute.
Clinical training: The appointee is expected to be a specialist registrar with a large interest in cancer, likely a medical
or clinical oncology trainee although the opportunity does not need to be restricted to these specialties if the
appointee is truly interested in this field of research. The appointee will be expected to take an integral role in the
planned trials of parental ColoAd1. In doing so they would be supervised by Dr Rachel Midgley but would take the
major responsibility for running the studies and would attend the appropriate clinics (likely colorectal oncology and
possibly colorectal surgical). They would receive a broader base training around colorectal cancer from Dr Midgley
within clinics but also by attending the MDTs and becoming an integral part of the team, and they would become a
very active contributor to colorectal cancer research both within and outwith Oxford. It is expected that this project
would be looked at favourably by the Royal Colleges and would count towards the final training goals of the
appointee. He/she will also develop a good grasp of GCP and of the processes involved in setting up, running and
authoring publications about complex clinical trials.
Tumour mutations identified by whole genome sequencing as predictive markers for progression of high risk
(T1G3) bladder cancer.
Dr Anne Kiltie (scientific/clinical; lead supervisor)
Prof Freddie Hamdy (scientific/clinical)
Professor Ian Tomlinson (scientific/clinical)
Background
Patients with T1G3 transitional cell carcinoma of the bladder who fail BCG treatment present a difficult challenge to
uro-oncologists. As radiotherapy is ineffective, radical surgical treatment with cystectomy is generally recommended
to prevent progression to the more lethal muscle invasive form of the disease. However, two forms of T1G3 tumours
exist, arising from either hyperplasia/high grade Ta disease (with few molecular alterations, i.e. low genomic
instability) or dysplasia/carcinoma in situ (with many more genetic alterations, Knowles, Int J Clin Oncol 13:287-97,
2008), so cystectomy may be overtreatment in a proportion of these individuals. Clinico-pathological features cannot
determine with any accuracy which patients’ tumours will progress.
In collaboration with the Wellcome Trust Centre for Human Genetics, Oxford, we have recently undertaken whole
genome sequencing (WGS) of DNA from 15 bladder tumour samples and matched germline DNA, in five papillary low
grade (Ta) tumours, five high grade non-muscle invasive T1G3 tumours and five muscle invasive tumours. We have
identified novel mutations in some muscle invasive and T1G3 tumours which are not present in Ta tumours; these
may be driver mutations for bladder cancer progression, and we hypothesise that these may also determine which
T1G3 tumours progress to muscle invasive disease. Therefore, these mutations may be useful as biomarkers in
determining which patients require cystectomy, and also may allow identification of suitable drug targets, for
prevention of tumour recurrence in patients at low risk of progression.
Project proposal
We shall select up to five genes from our WGS project, with mutations seen in muscle invasive and T1G3 disease but
not Ta tumours. These genes will then be sequenced in a larger set of bladder tumours to identify further mutations
and to determine their frequencies, using cutting edge methodology. Bioinformatic prediction software will be used
to identify putatively functional mutations, then functional assays will be performed in bladder cancer cell lines, to
study to the impact of mutations on cellular functions including proliferation, invasion, and other gene-specific
outputs. Promising candidates will then be taken forward to in vivo models, where efficacy of drug treatments which
target the relevant genes will also be tested where appropriate. As a major aim of the project is the preclinical
development of a panel of clinically useful biomarkers, the proteins expressed by the genes of interest shall also be
studied by immuno-histochemistry in archived paraffin-embedded T1G3 bladder tumour samples with known
outcome data, using our state of the art staining and imaging equipment, and correlated with progression.
Clinical/Translational relevance of project
Validation of mutations found in bladder tumours by whole genome sequencing could lead to the development of
prognostic clinical biomarkers in high risk non-muscle invasive bladder cancer. This could lead to prompt treatment
of potentially life-threatening disease and the sparing of low-risk individuals from over treatment, resulting in
improved cure rates with minimal toxicity. We may also be able to identify drugs suitable for intravesical
administration in low-risk patients.
Planned training and mentoring arrangements
Training will be provided on all aspects of the project by Dr Kiltie, Prof Tomlinson, Prof Hamdy and their laboratory
and biobanking teams. The fellow will attend weekly laboratory meetings/journal club to discuss the project with the
other members of Dr Kiltie’s group and to present their work formally at least monthly, and to be exposed to other
laboratory members’ projects. The fellow will also be expected to attend departmental internal and external
seminars regularly, both in the Gray Institute and the Wellcome Trust Centre. Mentoring will be provided for surgical
trainees through the Nuffield Department of Surgical Sciences and through the Department of Oncology for oncology
trainees, with appropriate clinical and scientific mentors. The fellow will also have the opportunity to attend one
clinic per week.
Radiation-related Normal Tissue Complications after Cancer Cure
Sarah Darby, PhD & Paul McGale, PhD (Scientific Supervisors)
Carolyn Taylor, FRCR (Clinical Supervisor)
Abstract
This project will consist of exploring and exploiting the possibilities provided by the recent widespread use of 3D CTbased radiotherapy treatment planning for estimating the long-term side effects of radiotherapy. It will consist
partly of extending the work carried out to date in our group on the risk of radiation-related heart disease after
radiotherapy for breast cancer, lymphoma, and childhood cancer, and partly of initiating work in new areas, such as
the long-term side effects of pelvic radiotherapy.
Project proposal
Around 3% of the UK population are cancer survivors, with the total number now around 2 million and increasing by
3% each year. The increasing trend is partly the consequence of an ageing population, but it also arises from
increasingly successful cancer treatments. Success in curing cancer is, however, not without its problems and cancer
survivors are at risk of increased morbidity and mortality from other diseases occurring as side-effects from their
treatment. It is now recognised, particularly in cancers with good prognosis, that cure alone is not enough and that
cancer survivorship represents a second stage of the cancer battle. This fact was recognised by the Cancer Reform
Strategy that created the National Cancer Survivorship Initiative (see http://www.ncsi.org.uk/).
Treating cancer usually involves balancing the benefits from a particular therapy against the risks and side effects.
Around two thirds of all cancer patients receive radiotherapy treatment. There is substantial information on early
side-effects of radiotherapy, as patients present with them while on treatment and in follow-up clinics. In contrast,
late side-effects, and especially those that mostly occur more than five years after radiotherapy, are less well
studied. Some insight into the long term side-effects of radiotherapy is provided by randomised clinical trials, but
only a small proportion of cancer patients are entered into a trial and follow-up is often less than 10 years. In any
case the data collected as part of the trial is not necessarily stored and collated in an appropriate way for studying
the late side-effects of radiotherapy.
Dedicated studies are therefore required to understand the frequency and extent of the long-term consequences of
radiotherapy for cancer. Careful study design, good outcome data and accurate dosimetry are all needed to enable
information about the experience of patients who have been irradiated in the past to be turned into useful estimates
of the long-term risks for patients being irradiated today. Over the past 10-15 years, opportunities for accurate
radiation dosimetric measurements on patients irradiated for cancer have increased rapidly due to the widespread
use of CT-based radiotherapy planning. This means that it is now possible to derive accurate estimates of the
radiation dose to specific normal tissues.
This project will consist of exploring and exploiting the potential provided by the huge amount of data being
produced by modern radiotherapy planning. It will consist partly of extending the work carried out to date in our
group on the risk of radiation-related heart disease after treatment for breast cancer, lymphoma, and childhood
cancer, and partly of initiating work in new areas, such as the long-term side effects of pelvic radiotherapy.
Clinical/ Translational relevance of project
The project will derive dose-response relationships for radiation-induced late effects. Our work to date has shown
that such dose-response relationships can be successfully developed, and are clinically extremely useful. Our team
have recently derived a dose-response relationship for the risk of heart disease following radiotherapy for breast
cancer. Breast cancer radiotherapy can reduce the risk of breast cancer recurrence and death. However, it can
increase the risk of heart disease. In the past, it has been difficult for Oncologists to weigh up the risks and the
benefits of radiotherapy. Our recent dose-response relationship for radiation-induced heart disease now enables
clinicians to do this. Estimates arising from our study are now starting to be used in clinical practice and also to
influence guidelines. For example, The Royal College of Radiologists have recently requested a summary of our work
to place on their website.
Accurate information regarding the risks of many other important late normal tissue complications of radiotherapy is
sparse. Modern radiotherapy techniques, such as intensity modulated radiotherapy (IMRT), Dynamic Arc Therapy
(e.g. RapidArc) and Stereotactic Body Radiotherapy (SBRT) give Oncologists unprecedented control over the delivery
of dose. The long-term aim of this work is to use detailed dosimetry and reliable outcome data to provide doseresponse relationships for various clinical endpoints.
The project will be based at the Clinical Trial Service Unit (CTSU) on Oxford University’s Old Road Campus, directly
opposite the new state-of-the-art Oxford Cancer Centre and next door to the Gray Institute for Radiation Oncology
and Biology. CTSU is internationally recognised as a centre of excellence for large scale clinical trials and
epidemiological research and is the home of the secretariat for the Early Breast Cancer Trialists’ Collaborative Group
(EBCTCG). Further details about CTSU are available on website: http://www.ctsu.ox.ac.uk. CTSU provides an
exceptional educational environment with expert individual supervision and support from a large number of
experienced and enthusiastic researchers with backgrounds in clinical medicine, statistics, and epidemiology. The
project supervisors are based in CTSU and will meet with the Fellow at regular intervals. In additional, there are
ample opportunities to attend seminars in clinical and academic oncology as well as in statistics and epidemiology.