25 June 2014 Annual Scientific Meeting Pavilions of Harrogate

Transcription

th
25 June 2014
Annual Scientific Meeting
Pavilions of Harrogate
PROGRAMME
PROGRAMME
2014 YCR ANNUAL SCIENTIFIC MEETING
PAVILIONS OF HARROGATE
Wednesday 25th JUNE
8.45 am
Registration and Coffee
9.30 am
Welcome – Professor Tony Robards
9.35 am
YCR Update – Charles Rowett
10.00 am
Open Papers - Scientific Session 1
11.15 am
Coffee, Poster Session and Scientific Exhibition
11.45 am
Open Papers – Scientific Session 2
12.45 pm
Buffet Lunch and Scientific Exhibition
1.00 pm
Poster viewing
2.15 pm
Open Papers – Scientific Session 3
3.30 pm
Tea, Poster Session & Scientific Exhibition
4.00 pm
Host: Professor Laurence Patterson
Guest Lecturer - Professor V Craig Jordan,
Scientific Director Lombardi Comprehensive Cancer Center,
Georgetown University Medical Center
Title of talk "Why It Is The Right Thing To Do: Invest In Young Cancer
Research Scientists!"
5.00 pm
Announcement of Poster Awards
5.05 pm
Closing address – Professor Tony Robards
5.30 pm
Drinks and canapés
The Open Papers and Guest Lecture will be held in the Aire Room,
Scientific presentations are 15 minutes duration and the
Guest Lecture is 1 hour including questions.
The Poster presentations, refreshments and Scientific Exhibition will be held in the Wharfe Room.
Please stand by your Posters from 1pm to 2.15pm and remove them from the stands at the end of the
afternoon Poster Session.
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Exhibitors
YCR would like to thank the support given from Exhibitors who are listed below!
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OPEN PAPERS – SCIENTIFIC SESSION 1
Chair: Professor Mohammad Ilyas
10.00 am
Genomic Copy Number Data from Next-generation Sequencing Predicts Bone
Metastasis in Breast Cancer
Henry M Wood1 , R E Coleman 2, W Gregory1, J Westbrook1, P Rabbitts1,
and JE Brown2
10.15 am
1
University of Leeds
2
University of Sheffield
Deciphering a Molecular Signature of Glioma Progression using microRNA Expression
Data
Josie Hayes, Alastair Droop, Helene Thygesen, Marjorie Boissinot, David Westhead,
Susan Short and Sean Lawler
Leeds Institute of Molecular medicine, University of Leeds.
10.30 am
Identifying Modifiers of MCPH1 induced Premature Chromosome Condensation
using an siRNA Screen
Victoria J. Cookson1, A Awaji1, M Adams2, J Higgins2, D Tomlinson2, J Bond1,
EE Morrison1 and SM Bell1
1
Leeds Institute of Biomedical and Clinical Sciences and 2The Bioscreening Technology
Group (BSTG), Wellcome Trust Brenner Building, St James’s University Hospital, Leeds
LS9 7TF.
10.45 am
Functional assembly of recombinant human MCM complex in isolated nuclei
Emma L.Hesketh1, D. Coverley1 and J.P. J. Chong1
1
11.00 am
Department of Biology, University of York, York, YO10 5DD.
The Role of the Tumour Microenvironment in HPV Positive and Negative
Oropharyngeal Carcinoma
Robert Bolt1, Bernadette Foran1, Daniel W Lambert1 and Keith D Hunter1
University of Sheffield, Sheffield Teaching Hospitals NHS Trust and Weston Park
Hospital, Sheffield.
11.15 AM – COFFEE
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Chair: Professor John Saxton
11.45 am
The YCR Centre for Early Phase Clinical Trials
Julia Brown, S Brown, C Twelves, F Collinson, D Sebag Montefiore,
R Coleman, P Woll, S Danson, M Lind and N Maitland
University of Leeds, University of Sheffield, University of Hull, University of York.
12.00 pm
A feasibility study testing four hypotheses with phase II outcomes in advanced
colorectal cancer (MRC FOCUS 3): A paradigm for randomised controlled trials in the
era of personalised medicine?
Susan D Richman, Gemma Hemmings, Phil Chambers, Morag Taylor,
Matthew Seymour and Phil Quirke on behalf of the FOCUS3 TMG.
Leeds Institute of Cancer and Pathology, St James’s University Hospital, Leeds LS9 7TF
12.15 pm
MRI and Ultrasound: A Synergistic Approach to the Localisation and Treatment of
Malignancy
Nina Louise Purvis, Dr Peter Gibbs, Dr Martin Pickles and Professor Lindsay Turnbull.
Centre for MR Investigations (HYMS at University of Hull), Hull Royal Infirmary, Anlaby
Road, Hull, HU3 2JZ
12.30 pm
A Systematic Review of the Literature Examining Inequalities in Access to Specialist
Palliative Care for People with Cancer
Donald J Nicolson, Hong Chen, Victoria Allgar, Una Macleod and Miriam Johnson.
University of Hull, Cottingham Road, Hull, HU6 7RX.
12.45 pm LUNCH
1.00 – 2.15 POSTERS
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Chair: Dr David Gilham
2.15 pm
The Biological Effects of FGFR3 Fusion Proteins
Sarah V Williams, L Wang, CD Hurst and MA Knowles.
Section of Experimental Oncology, Leeds Institute of Cancer and Pathology,
St James’s University Hospital, Leeds, LS9 7TF.
2.30 pm
Oncolytic virotherapy for Paediatric High Grade Glioma; Evaluation of the Effects of
Oncolytic Virus on Cell Viability, Migration and Invasion
Julia V Cockle1, Elizabeth Ilett1, Karen J Scott1, Anke Brüning-Richardson1, Susan
Picton2, Susan Short1 and Alan Melcher1
1
Leeds Institute of Cancer Studies and Pathology, St. James’s Hospital, Leeds University
LS9 7TF.
2.45 pm
Reovirus activation of NK Cells Increases the Efficacy of Rituximab for the Treatment
of CLL
Fiona Errington-Mais1, L. Ilett1, C. Parrish1, P. Hillmen1, and A. Melcher1
1
Leeds Institute of Cancer Studies and Pathology, St. James’s Hospital, Leeds University
LS9 7TF.
3.00 pm
New Strategies for the Development of Integrin Antagonists as Improved Anticancer
Therapeutics
Adam Throup, Mark Sutherland, Andrew Gordon, Fatemah Al-Shammari, Hanadi
Ahmedah, Manar Zraikat, Steven D Shnyder, Laurence H Patterson and
Helen M Sheldrake.
Institute of Cancer Therapeutics, University of Bradford, Bradford, BD7 1DP.
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3.15 pm
FANCD2 Expression correlates with Glioma Grade and Chemical Inhibition of the
Fanconi Anaemia Pathway Sensitises Gliomas to Chemotherapeutic Agents
Abhijit A Patil1, Parag Sayal2, Marie-Lise Depondt1, Ryan D. D. Beveridge1, Anthony
Roylance2, Deepti H. Kriplani1, Katie N. Myers1, Angela Cox1, David Jellinek2, Malee
Fernando3, Thomas A. Carroll2 and Spencer J. Collis1*.
1
Sheffield Cancer Research Centre, Academic Unit of Molecular Oncology, Department
of Oncology, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10
2RX.
2
Neuro-Oncology Group, Sheffield Teaching Hospitals NHS Foundation Trust, Royal
Hallamshire Hospital, Sheffield S10 2JF.
3
Department of Histopathology, Sheffield Teaching Hospitals, Royal Hallamshire
Hospital, Sheffield S10 2JF.
3.30 – Coffee
4.00 – GUEST SPEAKER’S TALK Professor V. Craig Jordan
5.00 – 5.05 – Poster Awards
5.05 – 5.30 – Closing speeches
5.30 – Canapés & Drinks
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ORAL
PRESENTATIONS
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10.00am Talk – Henry Wood
Genomic copy number data from next-generation sequencing predicts bone metastasis in
breast cancer.
1
2
1
1
1
2
Wood HM , Coleman RE , Gregory W , Westbrook J , Rabbitts P , Brown JE .
1
University of Leeds
2
University of Sheffield
Being able to predict risk and likely site of metastasis would have huge implications for the clinical
management of patients. In breast cancer, bone metastasis is particularly common and is
characterised by a relatively long period prior to relapse.
We have looked for genomic differences in the primary tumours of three groups of patients: patients
who did not metastasise; patients who developed a bone metastasis; and other metastatic patients.
We used low coverage next-generation sequencing to generate genomic copy number data for 181
patients.
We analysed the differences between the groups in three ways. Inspection of common regions of
gain and loss showed no differences between the groups. However, a previously published
algorithm measuring local regions of genomic disruption did separate the groups, as did a novel
logistic regression algorithm that produces a genomic signature of prediction.
Using a combination of these algorithms, we able to make good predictions about the chance of
metastasis, and the likely site, simply by analysing tissue from the primary tumour.
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10.15 am Talk – Josie Hayes
Deciphering a molecular signature of glioma progression using microRNA expression data
JOSIE HAYES, Alastair Droop, Helene Thygesen, Marjorie Boissinot, David Westhead, Susan
Short, Sean Lawler
Leeds Institute of Molecular medicine, University of Leeds
Involvement of microRNAs in glioblastoma biology (GBM) has been well documented but less is known
about microRNAs involved in lower grade glioma. This study used microRNA expression data from The
Cancer Genome Atlas to identify microRNAs associated with survival in grade III (anaplastic
astrocytoma) and grade IV (GBM) tumours.
Using penalized regression, nine microRNAs were identified as predictors of survival in GBM. In the
anaplastic astrocytoma group, comparison of microRNA sequencing data from patient groups (n=482)
representing the extremes of survival identified 35 differentially expressed microRNAs.
Five microRNAs; miR-34a, miR-148a, miR-222, miR-9 and miR-182, were negatively associated with
prognosis in both GBM and anaplastic astrocytoma. Four microRNAs were associated with survival in
GBM but not astrocytoma; miR-145, miR-370, miR-10b and miR-124a. Of these four, miR-10b and miR124a have previously been reported to be involved in proliferation, migration and invasion in GBM and
also showed altered expression patterns in GBM (n=482) versus normal brain (n=10).
In conclusion, miR-34a, miR-148a, miR-222, miR-9 and miR-182, are associated with prognosis in both
grade III and IV gliomas. These microRNAs may represent a ‘signature’ for prognosis and their levels
may be exploited for predicting whether progression is imminent.
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10.30am Talk – Victoria Cookson
Identifying Modifiers of MCPH1 induced Premature Chromosome Condensation using an
siRNA Screen
1
1
2
2
2
1
1
VJ COOKSON , A Awaji , M Adams , J Higgins , D Tomlinson , J Bond , EE Morrison and SM
1
Bell
1
2
Leeds Institute of Biomedical and Clinical Sciences and The Bioscreening Technology Group
(BSTG), Wellcome Trust Brenner Building, St James’s University Hospital, Leeds LS9 7TF, United
Kingdom
Reduced levels of MCPH1 in breast (29%) and ovarian (19%) cancer are associated with increasing
tumour grade and poor survival. Loss of MCPH1 also causes premature chromosome condensation
(PCC). We have performed a human druggable genome siRNA screen to identify modifiers of PCC
and synthetic lethality in MCPH1 deficient cells. This should allow new drugs to be developed for
women with breast and ovarian cancers resistant to current chemotherapies.
MCPH1 siRNA knockdown was performed in U-2 OS cells to induce PCC. The siRNA screen was
carried out using Dharmacon siGENOME® SMARTpool® siRNA Libraries. Images were captured
using the Operetta high content imaging system and analysis carried out using Columbus
TM
(Perkin
Elmer). Hits were determined by z score calculations using a cut off value of +/- 2 SD and validation
was performed using deconvoluted siRNAs and Western blotting.
From the human Protein Kinase sub-library, we have identified 3 novel modifiers of MCPH1
function; PIK3R4, PACE-1 and MYT1. Interestingly, we have shown that MCPH1 regulates the
expression of the threonine/tyrosine kinases, MYT1 and WEE1, which control mitotic entry via
inhibitory phosphorylation on CDK1. The WEE1 inhibitor, MK1775, is currently undergoing Phase II
clinical trials and may prove as an effective therapy for women with aggressive cancers expressing
low levels of MCPH1.
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10.45am Talk – Emma Hesketh
Functional assembly of recombinant human MCM complex in isolated nuclei.
1
1
1
E. L. HESKETH , D. Coverley and J.P. J. Chong .
1
Department of Biology, University of York, York, YO10 5DD, UK.
DNA replication is a tightly regulated process ensuring chromosomal DNA is only replicated once
per cell cycle. In eukaryotes, six mini-chromosome maintenance (MCM) proteins form a complex
that provides DNA unwinding activity that is essential for replication fork progression.
Using recombinant hMCM that we have purified and verified using a set of biochemical assays, and
a cell free system derived from mammalian cells we are unravelling the steps required for the
functional assembly of the hMCM complex inside nuclei. Our focus is on how hMCM complex
assembly and activity is controlled by sequential phosphorylation by cellular kinases that are
commonly mis-expressed in cancer cells. We have recapitulated time-dependent assembly of
recombinant hMCM in mid-G1 phase nuclei and find that recombinant hMCM preferentially binds to
chromatin in the presence of cyclin E/cdk2, which in turn affects the activity of hMCM.
Once the functional assembly of hMCM is understood we hope to identify how this process is
corrupted in some cancer cells, generating information that can be used to design novel cancer
therapies.
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11.00am Talk – Robert Bolt
The Role of the Tumour Microenvironment in HPV Positive and Negative Oropharyngeal
Carcinoma
ROBERT BOLT, Bernadette Foran, Daniel W Lambert, Keith D Hunter
University of Sheffield, Sheffield Teaching Hospitals NHS Trust and Weston Park Hospital, Sheffield
UK
We present in-vitro evidence that altered tumour-stromal interplay offers a route through which
survival benefit may be imparted to HPV-positive disease. Conditioned media were collected from
HPV-positive and HPV-negative cell lines, and incubated with normal oral fibroblasts. Further
conditioned media were then collected from the stimulated fibroblasts and used in the experiments.
Contemporary “scratch” assays were undertaken using a “silicone stopper” technique to assess the
influence of conditioned media on cell migration. Proliferation was assessed through the MTS
assay. HPV-negative cell lines demonstrated a marked increase in void closure over 48hrs in
response to conditioned media when compared to control (P<0.01). Conversely, HPV-positive cell
lines demonstrated no significant change in void closure when compared to control. No significant
difference in proliferation was observed when comparing the effect of conditioned media on HPVpositive versus HPV-negative cell lines (P>0.05). Our data supports a marked difference in tumourstromal interaction between HPV-positive and -negative HNSCC; we further discuss the
mechanisms behind the differences observed.
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11.45am Talk – Julia Brown
The YCR Centre for Early Phase Clinical Trials
J BROWN, S Brown, C Twelves, F Collinson, D Sebag Montefiore, R Coleman, P Woll, S Danson,
M Lind, N Maitland
University of Leeds, University of Sheffield, University of Hull, University of York
The YCR ECTN has been funded to deliver a portfolio of early clinical trials across Yorkshire to
bridge the gap between basic laboratory research and the clinic. A multidisciplinary network of
Leeds/Bradford, Sheffield, and Hull/York cancer centre researchers and methodological/clinical trial
expertise from the Leeds Clinical Trials Research Unit will provide a framework for the delivery of
early clinical trials, with expedited trial proposal development, set-up and delivery to time/target. The
Network will support researchers to develop their ideas, facilitating high quality grant applications,
and providing ongoing mentorship. Key areas for research include:

immunological approaches, including viral therapy;

combining targeted systemic therapies;

new technical advances in surgical practice;

novel approaches to drug delivery;

studies tailored to specific under-represented and/or rare populations;

novel radiation techniques to reduce toxicity or improve efficacy.
The YCR ECTN will provide the opportunity for more Yorkshire-based patients to participate in
innovative early clinical trials, enabling earlier access to novel therapies and more targeted
treatments.
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12.00pm Talk – Susan Richman
A feasibility study testing four hypotheses with phase II outcomes in advanced colorectal cancer
(MRC FOCUS 3): A paradigm for randomised controlled trials in the era of personalised
medicine?
SUSAN D RICHMAN, Gemma Hemmings, Phil Chambers, Morag Taylor, Matthew Seymour and Phil
Quirke on behalf of the FOCUS3 TMG.
All authors from Leeds Institute of Cancer and Pathology.
Purpose: Molecular characteristics of cancer vary between individuals. In the future, most trials will
require assessment of biomarkers to allocate patients into enriched populations in which targeted
therapies are more likely to be effective. The MRC FOCUS 3 trial is a feasibility study to assess key
elements in the planning of such studies in a multicentre setting in the UK.
Patients and Methods: Patients with advanced colorectal cancer were registered from 24 centres
between February 2010 and April 2011. Following patient consent, formalin-fixed, paraffin
embedded (FFPE) tumour samples were sent to laboratories in Cardiff or Leeds for analysis of
KRAS/BRAF mutation status and topoisomerase 1 immunohistochemistry. Patients were then
classified into 1 of 4 molecular strata, which determined the set of 2 hypothesis-driven experimental
therapies they could be randomised to, all being compared to control chemotherapy (irinotecan, 5FU and folinic acid [LV5FU]). Patient information sheets (PIS) were used to avoid patient overload.
Results: 332 patients were registered and 244 randomised. 85% of eligible patients gave consent
to randomisation. Among randomised patients, biomarker results were provided within 10 working
days (wd) in 71%, 15 wd in 91% and 20 wd in 99% patients. KRAS mutation was detected in 88
(36%), BRAF in 15 (6%). 77% of patients were high (2-3), 19% low (0-1) and 4% inconclusive for
topo-1 expression. DNA mutation analysis was 100% concordant between two laboratories. Over
90% of participants reported that they either fully or mostly understood all aspects of the trial. In this
randomised phase II setting, omission of irinotecan in the low topo-1 group was associated with
increased response rate, and addition of Cetuximab in the KRAS, BRAF wildtype cohort was
associated with longer progression free survival.
Conclusion: Patient samples can be collected and analysed at designated reference laboratories
within clinically workable timeframes and with reproducible mutation results. Complex multi-arm
designs can be made acceptable to patients through good PIS, ensured by patient and carer input
into their design. Randomisation within each cohort provides the basis for outcome data that can
inform clinical practice. This work has led to the £5million funding by EME of the FOCUS4 trial.
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12.15pm Talk – Nina Purvis
MRI & Ultrasound: A synergistic approach to the localisation and treatment of malignancy
1
1
1
MISS NINA LOUISE PURVIS , Dr Peter Gibbs , Dr Martin Pickles , Professor Lindsay Turnbull
1
1
Centre for MR Investigations (HYMS at University of Hull), Hull Royal Infirmary, Anlaby Road, Hull,
HU3 2JZ
Positive tumour margin status remains a significant problem in breast conserving surgery often
resulting in reoperation. Breast tumours are being diagnosed earlier due to improved imaging
modalities and screening programs, meaning tumours may be small and occult. MRI provides
excellent delineation of a breast tumour’s extent but translating this information into surgical practise
is challenging. Magnetic Resonance-guided Focused Ultrasound (MRgFUS) is a non-invasive
surgical technology that allows thermal ablation of tissue deep inside the body without harming
surrounding tissue. By utilizing MRgFUS to create a series of thermally induced ‘lesion tattoos’ on
the periphery of a tumour it is hoped the increased tissue stiffness produced, resulting in improved
tumour palpation, will facilitate improved surgical outcome. In-house software has been developed
to plan lesion tattoos using MR images and this software has been applied to an ex vivo turkey
breast model to assess the software by comparing pre-ablation MR images, planned sonication
locations, post-ablation MR images and dissected turkey breast. It was found that the software
successfully allowed for the planning and implementation of thermal ablations in their required
location. A change in tissue colour and stiffness was also observed. Future work concerning lesion
tattooing will develop a breast tumour model using ex-vivo sheep mammary tissue in order to
generate data to support proceeding to human studies.
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12.30pm Talk – Donald Nicolson
A systematic review of the literature examining inequalities in access to specialist palliative
care for people with cancer
DONALD J NICOLSON, Hong Chen, Victoria Allgar, Una Macleod and Miriam Johnson
University of Hull, Cottingham Road, Hull, HU6 7RX.
BACKGROUND
Specialist Palliative Care (SPC) prevents and relieves the pain that people who may be dying feel.
Unequal access to SPC services, for people with persistent or complex palliative needs, is a
concern, and people from deprived backgrounds have poorer access than more affluent people.
Studies have found SPC varies with age, sex, diagnosis, ethnicity and socio-economic status
(SES). We are conducting a systematic review to examine the relationship between SES /
demographic variables, access to SPC, and place of death for patients with cancer.
METHODS
We searched 5 databases using standard systematic literature review methods. We included
studies of any design with adults diagnosed with or treated for cancer, which examined access to
SES/demographic variables, SPC and place of death. Two researchers checked 7,000 references
by title and abstract, and then full papers for possible inclusion.
RESULTS AND CONCLUSIONS
This is work in progress and we will present the results at the conference. We included 53 studies to
be quality assessed and data extracted. The added value of our study is that it will provide clearer
understanding of the relationship between access to specialist palliative care services and socioeconomic status, and their influence on where people with cancer die.
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2.15pm Talk – Sarah Williams
Biological Effects of FGFR3 Fusion Proteins.
WILLIAMS SV, Wang L, Hurst CD, Knowles MA.
Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James’s University
Hospital, Leeds, LS9 7TF.
We were the first to report the discovery of fusion genes involving FGFR3 in a proportion of bladder
cancers and cell lines last year. This discovery was particularly exciting in view of the observation
that cells containing the fusion genes are very sensitive to FGFR inhibitor drugs including some
currently involved in clinical trials.
We have investigated the mechanisms through which these fusion genes may exert their effects.
When expressed in NIH-3T3 cells the fusion genes show high levels of constitutive FGFR3
phosphorylation, equaling or exceeding the levels shown by FGF1 stimulated wild type FGFR3, and
the cells show a transformed phenotype. Expression of the partner genes, TACC3 and BAIAP2L1,
in NIH-3T3 cells does not lead to morphological transformation.
We have made some artificial gene constructs to investigate this further. We show that while loss of
the terminal exon of FGFR3 is universal in the fusion genes, it is not sufficient to account for the
oncogenic effects. Constitutive dimerisation may be important.
In addition to bladder cancer, FGFR3 fusion genes have now been reported in some other cancers
including glioblastoma and squamous lung cancer. They are likely to be a useful target for
personalised medicine across these cancers.
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2.30pm Talk – Julia Cockle
Oncolytic virotherapy for paediatric high grade glioma; Evaluation of the effects of oncolytic
virus on cell viability, migration and invasion
1
1
1
1
2
JULIA V COCKLE , Elizabeth Ilett , Karen J Scott , Anke Brüning-Richardson , Susan Picton ,
1
Susan Short , Alan Melcher
1
1
Leeds Institute of Cancer Studies and Pathology, St. James’s Hospital, Leeds University LS9 7TF,
United Kingdom
2
Yorkshire Regional Centre for Paediatric Oncology and Haematology, Leeds General Infirmary,
Great George Street, Leeds, LS1 3EX, United Kingdom
Paediatric high grade gliomas (pHGGs) are devastating tumours with dismal survival outcomes.
Oncolytic virotherapy uses viruses to selectively infect and destroy cancer cells, offering a novel
treatment approach. Here, we evaluate the in vitro cytotoxic effects of herpes-simplex virus (HSV),
measles, reovirus and vaccinia, on a panel of paediatric glioma cell lines. We describe, for the first
time, the effects of oncolytic viruses (OV) on the migratory behaviour of pHGG cells.
Cell viability was examined over 96hrs by MTT and FACS-based assays. Migratory behaviour was
examined using 2D scratch assays and 3D spheroid invasion assays in collagen. The sensitivity of
pHGG lines to OV was demonstrated by a loss of cell viability over 96 hours. The cytotoxic effect
was particularly marked for cell lines treated with vaccinia, while sensitivity to other viruses was cellline dependent. Analysis of migratory and invasion assays indicated that HSV at MOI 10 resulted in
a near total blockade of both migration at 24 hours, and invasion at 72 hours, in all cell lines tested.
Our results demonstrate that pHGG cells are sensitive to the cytolytic effect of a range of OV and
that HSV can block the migration and invasion of glioma cells in vitro.
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2.45pm Talk – Fiona Errington-Mais
Reovirus activation of NK cells increases the efficacy of rituximab for the treatment of CLL.
1
1
1
1
1
F. ERRINGTON-MAIS, L. Ilett, C. Parrish, P. Hillmen and A. Melcher
1
LICAP, St James’s University Hospital, Leeds. LS9 7TF
Chronic Lymphocytic Leukaemia (CLL) is associated with the accumulation of malignant B
lymphocytes and remains an incurable disease. Rituximab is a chimeric anti-CD20 monoclonal
antibody used for the treatment of CLL and it induces its cytotoxic effects by NK cell-mediated
antibody-dependent cellular cytotoxicity.
Reovirus exerts its anti-cancer activity by direct oncolysis or activation of innate and/or adaptive
anti-tumour immunity. Reovirus treatment of PBMCs increases NK-mediated lysis of tumour cell
targets, in vitro, and NK cell activation has also been observed in vivo after systemic delivery.
This study investigates the direct cytotoxic effects of reovirus against CLL and examines whether
reovirus could be used to increase the efficacy of rituximab-based immunotherapy.
These data show that CLL cells are susceptible to reovirus oncolysis. CLL cell lines are susceptible
to NK cell-mediated lysis and reovirus activation of PBMCs, increases lysis of CLL cell line targets.
Importantly, NK cells from CLL patients are activated by reovirus in the context of autologous
tumour cell targets. This study demonstrates that reovirus activation of PBMC could be used to
increase the efficacy of rituximab-based immunotherapy, providing a novel approach for the
treatment of CLL.
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3.00pm Talk – Helen Sheldrake
New Strategies for the Development of Integrin Antagonists as Improved Anticancer
Therapeutics
Adam Throup, Mark Sutherland, Andrew Gordon, Fatemah Al-Shammari, Hanadi Ahmedah, Manar
Zraikat, Steven D Shnyder, Laurence H Patterson, HELEN M SHELDRAKE
Institute of Cancer Therapeutics, University of Bradford, Bradford, BD7 1DP
The integrin family of cell surface receptors mediate cell-extracellular matrix interactions controlling
cell adhesion and transmembrane signalling involved in cell migration, proliferation, differentiation,
angiogenesis and apoptosis. Members of the RGD binding integrin subfamily are often upregulated
in cancers, notably melanoma, prostate adenocarcinoma, and glioblastoma. Because of their ability
to increase tumour proliferation and invasion, and control angiogenesis and metastasis, antagonism
of these receptors is an interesting therapeutic target, but identifying the optimum approach remains
a challenge.
We have identified several new strategies for integrin antagonism affording novel targeted therapies
for advanced cancers where there are currently few effective treatments available, and improved
efficacy and safety compared to existing compounds. The identification and characterisation of hit
compounds for further development into new medicines will be described.
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3.15pm Talk – Abhijit Patil
FANCD2 expression correlates with glioma grade and chemical inhibition of the Fanconi
Anaemia pathway sensitises gliomas to chemotherapeutic agents.
1
2
1
1
2
ABHIJIT A. PATIL , Parag Sayal , Marie-Lise Depondt , Ryan D. D. Beveridge , Anthony Roylance ,
1
1
1
2
3
Deepti H. Kriplani , Katie N. Myers , Angela Cox , David Jellinek , Malee Fernando , Thomas A.
2
1*
Carroll & Spencer J. Collis .
1
Sheffield Cancer Research Centre, Academic Unit of Molecular Oncology, Department of
Oncology, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.
2
Neuro-Oncology Group, Sheffield Teaching Hospitals NHS Foundation Trust, Royal Hallamshire
Hospital, Sheffield S10 2JF, UK
3
Department of Histopathology, Sheffield Teaching Hospitals, Royal Hallamshire Hospital, Sheffield
S10 2JF, UK
Abstract
Brain tumours kill more children and adults under 40 than any other cancer. Around half of primary
brain tumours are high-grade malignant astrocytomas, also known as glioblastoma multiforme
(GBMs). Treatment of GBM remains a significant challenge with a median patient survival of ~1yr.
Survival rates have improved little over the last 40 years highlighting an unmet need for the
development of novel targets and agents to improve GBM treatment. Previous work has suggested
that up-regulation of DNA response (DDR) pathways in GBM may contribute to their inherent and/or
acquired resistance to radiation and chemotherapeutic agents. Using archived and fresh glioma
tissue, we show that in contrast to normal brain or benign schwannomas, high-grade gliomas exhibit
elevated expression of FANCD2, a key protein of the Fanconi Anaemia (FA) DNA repair pathway.
Importantly, FANCD2 expression levels correlate with tumour grade. These data reveal a potential
therapeutic window to allow inhibition of the FA pathway in tumour cells, whilst sparing normal brain
tissue. Using several inhibitors of the FA pathway (FAi) in combination with FA-proficient and FAdeficient glioma cell lines and primary GBM cultures; we demonstrate that inhibition of the FA
pathway sensitises gliomas to the chemotherapeutic agents Temozolomide and Carmustine
irrespective of MGMT status. Our findings provide a strong rationale for the development of
novel/potent inhibitors of the FA pathway to improve the treatment of GBMs.
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POSTERS
PRESENTATIONS
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POSTER 1
Modulation of neuroblastoma polysialic acid biosynthesis inhibits tumour cell migration and
invasion
Robert A. Falconer*, VIRGINIE VIPREY, Bradley R. Springett, Yousef M.J. Al-Saraireh, Matthew
Northrop, Mark Sutherland, Rida Saeed, Paul M. Loadman, Steven D Shnyder and Laurence H.
Patterson
Institute of Cancer Therapeutics, School of Life Sciences, University of Bradford, West Yorkshire
BD7 1DP, U.K.
Tel: +44 (0)1274 235842; Email: [email protected]
Polysialic acid (polySia) is expressed on the surface of NCAM (neuronal cell adhesion molecule) in
many cancer cells where it modulates cell adhesion, migration, invasion and metastasis. It is
strongly associated with poor clinical prognosis and correlates with aggressive/invasive disease in
small cell lung cancer, neuroblastoma and many other tumours. SiRNA knockdown of
polysialyltransferase ST8SiaII, responsible for polySia synthesis in tumours, abolishes tumour cell
migration.
Using CMP as a tool compound, we have validated a highly sensitive HPLC-based inhibition assay,
and demonstrated competitive inhibition of ST8SiaII. CMP additionally causes a reduction in polySia
expression, tumour cell migration and invasion. We have synthesised inhibitors of ST8SiaII. Using
isogenic cell lines and naturally expressing human neuroblastoma cells (SH-SY5Y, IMR-32), these
compounds were evaluated for their ability to reduce polySia expression and to modulate cell
migration in vitro. We have identified CMP-sialic acid precursors, including compounds ICT-3172
and ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These
effects were only found in cell lines expressing ST8SiaII and polySia.
In summary, we have identified a number of key modifications to polySia biosynthetic precursors
which dramatically decrease cell migration in cells over-expressing ST8SiaII through modulation of
polySia assembly.
24 | P a g e
POSTER 2
Differentiation of neural crest derived peripheral neurons to model neurocristopathies
1
1
1
JIM HACKLAND , David Preskey , Christian Unger & Peter Andrews
1
1
Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield
The neural crest is a migratory tissue that plays a role in early development contributing to a wide
range of tissues throughout the body. Neuroblastoma is one of a group of tumours that are thought
to arise from the neural crest as a result of defects in the development of the tissue. Using human
ES and iPS cell lines, we have developed a robust protocol for the differentiation of human
pluripotent stem cells into putative neural crest progenitors. These cells express high levels of the
surface antigens p75 and HNK-1 when subjected to this protocol. We have shown that expression
of the neural crest stem cells markers SOX10 and AP2α segregate to the p75
High
population.
Following induction of neural crest we differentiated peripheral neurons, creating the cells in which
neurocristopathies, such as neuroblastoma, may originate.
In parallel to developing an efficient differentiation protocol, we have reprogrammed neuroblastoma
cells to neuroblastoma iPS (niPS) cells that contain a complete set of cancer specific genome
changes. The generation of neural crest derivatives, such as peripheral neurons, from niPS cells
now provides the opportunity to study the in vitro effect of genetic and epigenetic elements of
neuroblastoma during early human development.
25 | P a g e
POSTER 3
Radiomics Analysis of MRI data
M. LOWRY, L. Kenning and L.W. Turnbull, Centre for MR Investigations, University of Hull.
Genetics and tumour micro-environment affect glioma treatment response and survival.
We used radiomics to analyse multi-parametric MRI data for underlying relationships.
Morphological, diffusion tensor, multi-flip-angle T1, and dynamic T1 and T2* contrastenhanced 3.0T MRI was performed in 55 patients. Registered, processed parameter
volumes were combined into one 4D volume before statistical sampling. For cluster
analysis, the 16 derived parameters were standardised to their median distribution.
Dendrograms were constructed and formatted into a heat map.
The heat map was non-random (P<0.0001). Seven parameter clusters were recognised;
several parameters were correlated, showing redundancy in lesion differentiation. For
patient clusters, we identified two large high-grade-lesion clusters, one closely linked to a
low-grade-lesion cluster and with an intermediate parameter profile. Both high-grade
clusters showed similar proportions of deaths.
Multi-parametric MRI provides useful diagnostic and prognostic information; however, it
can produce an overwhelming amount of data, and identification of associations requires
more than simple statistical comparisons. Radiomics provides a framework for assessing
associations between parameters and disease characteristics. It demonstrated some
redundancy amongst our parameters but revealed ≥1 useful parameter for each MRI
paradigm. As a minimum set, we suggest Q, R1, Ktrans, rCBVb, FLAIR, ADC, K2.
Analysis of post-treatment parameter changes could predict survival.
26 | P a g e
POSTER 4
Targeting the Formyl-peptide receptor-1 for treatment of high grade glioma
1
1
1
1
1
1
2
D. S. AHMET, M. V. Vinader, F. Bajwa, Ilona Pysz, D. Healey, S. D. Shnyder, X. W. Bian, L.
1
1
H. Patterson, K.Afarinkia *
1
Institute of Cancer Therapeutics, University of Bradford, Tumbling Hill Street, BD7 1DP
2
Southwest Hospital, TMMU, Chongqing, People’s Republic of China
Glioma account for over half of all primary brain tumours and have a very poor prognosis, with a
median survival of less than two years. None of the therapeutic interventions currently used for
glioma are particularly successful & invariably fail to prevent recurrence. These difficulties are
associated with the aggressive nature of the high grade tumours. It has been identified that FPR-1
is a key player in driving the invasiveness and rapid rate of growth. Therefore, targeting FPR-1 with
small molecule antagonists could have therapeutic potential.
We have identified a number of FPR-1 antagonists. We show that these small molecule
antagonists reduce calcium mobilisation, proliferation and migration of the glioma cell line U87 MG.
We also began an investigation into the relationship between necrosis/ hypoxia and the expression
of FPR-1 on spheroid models.
27 | P a g e
POSTER 5
Prediction of progression free survival in high grade gliomas using pre-operative MR
1
2
2
3
LAWRENCE KENNING , Martin Lowry , Martin Pickles , Christopher Rowland-Hill , Shailendra
3
3
Achawal , Chittoor Rajaraman , and Lindsay W Turnbull
2,3
1
2
Centre for Magnetic Resonance Investigations, University of Hull, Centre for Magnetic Resonance
3
Investigations, Hull York Medical School, Hull and East Yorkshire Hospitals NHS Trust
Aim: To investigate whether pre-operative MR parameters could predict progression free survival at
210 days in high grade gliomas.
Methods: Multi-parametric MR data was acquired from 45 patients with histologically proven high
grade gliomas using a 3.0T scanner. Pre-operative morphological imaging was acquired along with
DTI, DCE and DSC. Parametric volumes were created by registering functional maps into a single
4D volume. Tumour volumes of interest were contoured using morphological imaging. Gaussian
mixture modelling (GMM) was applied to each parameter, generating a further two populations for
each parametric volume (TUM0 and TUM1). Kaplan-Meier survival analysis at 210 days from preoperative MR was calculated for all groups. Log rank tests were used to test for significant
differences.
Results: Nine of the 39 mean values were significant discriminators of progression free survival at
210 days using P<0.05. Twenty-three of 45 cases showed progression at 6 months.
Parameter
Grade
ADC
TUM0
q
TUM1
RD
TUM0
TUM
Ktrans
TUM0
TUM1
TUM
ve
TUM1
K2
TUM0
Median Cutoff
N
Events
Estimate
(Days)
95% Confidence Interval
Lower
Bound
Upper
Bound
>=
1.025
22
9
186
170
202
<
1.025
23
14
161
142
181
>=
0.457
22
16
159
142
176
<
0.457
23
7
189
171
207
>=
0.894
22
9
186
170
202
<
0.894
23
14
161
142
181
>=
0.069
22
14
164
145
182
<
0.069
23
9
183
165
201
>=
0.040
22
14
157
136
177
<
0.040
23
9
189
175
203
>=
0.122
22
15
156
136
176
<
0.122
23
8
190
176
204
>=
0.128
22
15
158
138
177
<
0.128
23
8
189
173
204
>=
0.189
22
14
163
145
182
<
0.189
23
9
183
165
201
>=
-1.698
22
7
185
167
202
<
-1.698
23
16
164
146
182
Sig.
0.041
0.002
0.041
0.040
0.012
0.005
0.007
0.034
0.016
Discussion: Functional parameters derived from pre-operative MR can predict progression free
survival at 210 days. The of use dynamic imaging to derive parameters such as K
trans
, ve and K2
should be considered for pre-operative MR assessment in these patients. Furthermore the use of
GMMs should be considered for the evaluation of heterogeneous populations. Functional MR could
subsequently identify patients who would benefit from a shorter scan interval or need to be scanned
earlier during treatment to identify early signs of progression.
28 | P a g e
POSTER 6
Identification and characterisation of microRNAs involved in glioblastoma
cell proliferation and survival
1
1
2
2
2
3
BOISSINOT M. , Hayes J. , Adams M. , Higgins J. , Tomlinson D. , Lawler S.E. , Short S.
4
1-Translational Neuro-Oncology Group, LICAP, University of Leeds, UK
2-BioScreening Technology Group, University of Leeds, UK
3-Dept of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA
4-St James’s Institute of Oncology and LICAP, University of Leeds, UK
Introduction: There is a pressing need for new therapeutic approaches for the treatment of
glioblastoma (GBM). MicroRNAs (miRs) are single-stranded non-coding RNAs that have been
shown to play roles in multiple hallmark characteristics of GBM, suggesting that miRs and their
associated pathways may be of therapeutic importance.
Methods: The miRIDIAN mimic library (Dharmacon) that encompasses all human miRs
annotated in miRBase v16.0 was used with a high-throughput imaging platform (Operetta) to
identify miRs with potent effects on GBM cell proliferation and survival. Screens were performed in
duplicate on U251 (adult) and KNS42 (paediatric) GBM cell lines. Cell number was assessed 72h
post-transfection and expressed as z-scores of the nuclei count. Validation included RTqPCR,
imaging and flow cytometry based assays.
Results: For each cell line, the functional screen resulted in approximately 100 candidates,
of which 70% were in both cell lines. Seven miRs were validated in a panel of 4 adult and 2
paediatric GBM cell lines. All candidates caused cell cycle arrest and one candidate caused
apoptosis across all cell lines.
Conclusion: We have identified seven microRNAs with potent effects on survival and
proliferation of GBM cell lines. Further validation is ongoing in order to further characterise the
mechanisms involved.
29 | P a g e
POSTER 7
Use of Photodynamic Therapy Drugs on Primary Prostate Epithelial Cells
1
2
2
FIONA M. FRAME , Huguette Savoie , Ross Boyle and Norman J. Maitland
1
1
YCR Cancer Research Unit, Department of Biology, University of York, Heslington, YO10 5DD.
Department of Chemistry, University of Hull, Cottingham Road, Hull, HU6 7RX.
2
Photodynamic therapy (PDT) is an established anti-cancer therapy that can be used on its own or in
combination with other therapies. PDT functions using a combination of a photosensitizer drug and
light; once activated by light the drug is cytotoxic. PDT has been successfully used for surface
tumours e.g. head and neck cancers, but its potential against internal tumours such as prostate
cancer has yet to be fully realized. This study explores the effect of a standard clinically used PDT
drug, Foscan, on primary prostate epithelial cells cultured from patient tissue (benign and
malignant). Further, we test a novel modified photosensitizer drug to which ligands can be
conjugated in order to target the drug to specific surface markers on cancer cells. Viability of cells
were measured using MTT assays. We found that primary cells were susceptible to the PDT drugs,
however they required increased concentration of drug compared to cell lines. In addition, we
observed variability between patient samples. Effect of the PDT drugs on prostate stem cells
(benign and malignant) using viability and clonogenic assays will also be explored.
30 | P a g e
POSTER 8
Patient-derived first generation xenografts of prostate cancers: promising tools for
predicting drug responses for personalised chemotherapy
1,2
1
Rambrakash Beekharry., M-C Labarthe-Last.,
2,3
2
4
1
M Simms, VM Mann, Chenglong Li, N J
1
Maitland., A T COLLINS.
1
YCR Cancer Research Unit, Department of Biology, University of York, York. YO10 5DD
2
Department of Urology, Castle Hill Hospital (Hull & East Yorkshire Hospitals NHS Trust),
Cottingham, HU16 5JQ,
3
Hull York Medical School, University of York, YO10 5DD.
4
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio, 43210, USA
The treatment of advanced prostate cancer remains disappointing and is hindered by the lack of
relevant preclinical models to improve development of effective therapeutic strategies. Early
passages of freshly generated xenografts are possibly better predictors of response and may better
represent the heterogeneity seen in prostate cancer than either cell lines or murine allografts. The
goal of this study was to determine if primary tumour xenografts represent the clinical heterogeneity
of human prostate cancer and whether the ability to form primary tumour xenografts is predictive of
clinical outcome and treatment response.
Tumour fragments from patients undergoing curative surgery or channel TURP for prostate cancer
were implanted into immunodeficient Rag2 C mice within 24 hours of surgery. Univariate and
-/-
-/-
multivariate analysis Cox proportional hazards models were used to assess the associations
between patient characteristics and clinical outcomes.
Tumour outgrowth was observed from 38 of 121 (31%) implanted prostate cancers, with 19 stable
lines generated. The ability to engraft correlated with advanced disease (r=0.22; P<0.05). Patients
who had undergone androgen ablation therapy were significantly more likely to engraft compared
with no-Xenograft patients (Odds ratio : 2.3, Relative Risk 58%, P< 0.003).
Patient derived xenografts may serve as important preclinical models. Tumours that engraft are
more aggressive and may be more representative of cancer with a higher propensity to relapse.
31 | P a g e
POSTER 9
Optimising Chromatin Immunoprecipitation to Unravel the Epigenetic Regulation of Latexin
and RARRES1 in Human Prostate Cancer Stem Cells
1
1
ALBERTO TAUROZZI , Davide Pellacani and Norman J. Maitland
1
1. YCR Cancer Research Unit, Department of Biology, University of York, York, YO10 5DD
Latexin and RARRES1 are the products of gene duplication on chromosome 3 and have a
complementary function in normal and malignant tissue homeostasis. The two genes are silenced in
stem cells, and expression is induced upon differentiation. Latexin and RARRES1 are also negative
regulators of self-renewal and metastasis, as demonstrated by reduced motility, invasion and
colony-forming potential when either gene is ectopically expressed within the stem cell
compartment.
Latexin and RARRES1 are regulated by DNA methylation in prostate cancer cell lines, but not in
primary disease. Our hypothesis is that in primary tissues, these genes are regulated by chromatin
status.
To investigate the relationship between histone modifications and gene expression in
heterogeneous tumours, we have established a Chromatin Immunoprecipitation (ChIP) protocol
capable of analysing low cell numbers (~500 cells). This will allow us to unravel the epigenetic
regulation of Latexin and RARRES1 in rare cancer stem cell (CSC) populations (<0.1% of tumour
cells) in direct comparison to their more differentiated progeny.
There is also the potential to investigate regulation through transcription factors such as the retinoic
acid receptor family which is known to regulate RARRES1 and has been implicated in the regulation
of Latexin.
32 | P a g e
POSTER 10
FOCUS4 Biomarkers: A cross-site pre-trial validation.
Susan D Richman, GEMMA HEMMINGS, Phil Chambers, Morag Taylor and Phil Quirke on behalf
of the FOCUS4 TMG.
All authors from Leeds Institute of Cancer and Pathology
Purpose: Molecular characterisation of tumours is leading to increasing personalisation of cancer
therapy, tailored to an individual and their cancer. FOCUS4 is a molecularly stratified clinical trial for
patients with advanced colorectal cancer. During an initial 16 week period of standard first-line
chemotherapy, consented tumour tissue will be tested for a variety of molecular assays, with the
results being used for randomisation. Laboratories in Leeds and Cardiff will carry out the molecular
testing. Prior to the trial opening, rigorous inter-laboratory validation was deemed necessary, to
ensure both laboratories could perform each molecular assay to the highest level of precision, and
demonstrate consistently high levels of concordance, as would be expected from laboratories
entrusted with centralised molecular testing.
Patients and methods: 97 FFPE tumour blocks were retrieved from the Wales Cancer Bank. Each
sample had ethical approval and consent for use in further bowel cancer research. Both laboratories
processed each sample in accordance with an agreed definitive and optimised FOCUS4 laboratory
protocol, and then reported the results to the MRC Trial Management Group (TMG) for independent
cross-referencing of the results.
Results: Pyrosequencing analysis of the mutation status at KRAS codons 12/13/61/146, NRAS
codons 12/13/61, BRAF codon 600 and PIK3CA exons 9 and 20, generated highly concordant
results. Only two samples gave discrepant results; in one case a PIK3CA mutation was detected
only in Leeds, and in the other, a PIKCA mutation was only detected in Cardiff. Tumour
heterogeneity was thought to be responsible for the pyrosequencing discrepancies. pTEN protein
expression and expression of the four mismatch repair (MMR) proteins was assessed by
immunohistochemistry (IHC) and resulted in 6/97 (6.2%) discordant results for pTEN and 6/388
(1.5%) for MMR IHC. Following joint review of the staining criteria by both labs, again full agreement
was reached for all tumours. Factors influencing discordances in IHC scoring included the presence
of signet ring cells, necrosis, mucin, edge artefact and over-counterstaining.
Conclusion: Both laboratories demonstrated the ability to carry out high standard molecular testing
of a large number of samples, with extremely high concordance rates (>94%), as expected from
centralised testing laboratories, ensuring confidence in their abilities to carry out reproducible
molecular testing throughout the course of the FOCUS4 clinical trial.
33 | P a g e
POSTER 11
Determining the effects of combining the PI3K inhibitor GDC-0941 with the vascular
disrupting agent combretastatin A-4-phosphate (CA4P) in colorectal carcinoma xenografts.
DEBAYAN MUKHERJEE, Jack E. Hurrell, Matthew Fisher, Andy W Brown, Gillian M. Tozer, Chryso
Kanthou
CR-UK/YCR Sheffield Cancer Research Centre, University of Sheffield, School of Medicine, Beech
Hill Road, Sheffield, S10 2RX.
Email: [email protected]
Vascular disrupting agents (VDAs) such as CA4P target tumours by stopping blood flow. However,
the tumour rim is relatively resistant to VDA treatment and consequently tumours re-grow from
within the viable rim. Previously we showed that nitric oxide production via endothelial nitric oxide
synthase (eNOS) confers resistance to CA4P. Here we investigate the pro-survival PI3K-AKTeNOS pathway and test whether the PI3K inhibitor GDC-0941 can increase the overall efficacy of
CA4P. SW1222 colorectal carcinoma xenograft blood vessels stained positive for eNOS. eNOS
levels were significantly higher in blood vessels in the periphery than those within the central
regions of the tumour. Treatment with CA4P (100 mg/kg, 24 h) induced significant central tumour
necrosis and loss of eNOS vessel staining. However, eNOS positive vessels within the viable
tumour rim and phosphorylation of AKT (pAKT) persisted within the rim. GDC-0941 (50 mg/kg) in
combination with CA4P induced similar levels of necrosis as CA4P but attenuated AKT
phosphorylation within the rim. GDC-0941 alone caused an increase in tumour apoptosis. Ongoing
studies are investigating the effects of combination treatment on tumour growth delay. Our results
suggest that the PI3K/-AKT-eNOS pathway may confer VDA resistance and therefore targeting this
pathway may improve the efficacy of VDAs.
34 | P a g e
POSTER 12
Three dimensional reconstruction of the anal sphincters to aid low rectal cancer surgery
WALKLETT C, Roberts N, Hale M, Magee D, Treanor D, Quirke P, West NP
All authors address: Leeds Institute of Cancer and Pathology Wellcome Trust Brenner Building,
St. James's University Hospital, Leeds LS9 7TF
Objective
We hypothesised that three dimensional (3D) histological reconstruction could offer unique insights
into the spatial anatomy of the rectum, nerves and surrounding structures leading to improved
surgical techniques.
Methods
Two human cadaveric adult pelvic exenteration specimens were obtained and underwent standard
histological analysis using whole mount serial sectioning, high resolution digital scanning and 3D
reconstruction. Digital slides were viewed and quantitated using Aperio ImageScope. 3D
reconstruction was performed with novel software using three consecutive blocks from one case at
1:10, 1:20 and 1:50 resolution.
Results
Tissue morphometry on digital slides demonstrated increasing skeletal muscle volume and
decreasing smooth muscle volume towards the anal aperture (p=0.001). Following 3D
reconstruction, to accurately view the structures, a minimum of 1:10 resolution was required. Minor
misalignment was identified between the blocks where tissue was lost during trimming therefore
further optimisation of the technique is required.
Conclusion
This novel study has established that 3D reconstruction of the low rectum is feasible to further
define the surgical anatomy of the anal sphincters and surrounding structures including nerves. We
believe that there is great potential to correlate this technique with preoperative MRI images to
study in-vivo anatomy and plan surgical roadmaps for future nerve sparing surgery.
35 | P a g e
POSTER 13
The Role of Metallothionein in Heavy Metal-Induced Urothelial Carcinogenesis
RHIANNON MCNEILL and Jennifer Southgate
Jack Birch Unit of Molecular Carcinogenesis, University of York
The development of bladder cancer is being increasingly associated with occupational exposure to
heavy metals such as cadmium. Heavy metals are only weakly mutagenic and thus their
mechanism of carcinogenesis remains unclear. The metallothioneins are proteins which bind to and
sequester heavy metals following exposure, but little is known about the specificity of induction or
the consequences of elevated metallothionein expression. The aim of this work was to examine the
metallothionein response in normal human urothelial (NHU) cells exposed to cadmium in vitro.
Twelve hours of cadmium exposure resulted in a striking induction of five metallothionein genes in
NHU cells and at 48 hours exposure, metallothionein protein expression was 5 times more
abundant in exposed cells. Discrimination between cadmium and its reactive oxygen species (ROS)
by-product found that only cadmium itself was capable of upregulating the metallothioneins.
Ongoing work aims to establish the longevity of metallothionein protein expression as a possible
biomarker of exposure and to investigate whether sequestration of cadmium within the cell provides
a mechanism for chronic exposure.
36 | P a g e
POSTER 14
A high-content approach to the identification of novel pro-survival protein kinases in
urothelial carcinoma.
HEATHER L MARTIN, Margaret Knowles and Darren C Tomlinson.
Leeds Institute of Biomedical & Clinical Sciences and School of Molecular and Cellular Biology,
University of Leeds, Leeds
Invasive urothelial carcinoma (UC) represents a significant proportion of patients diagnosed with
bladder cancer and has a poor 5-year survival rate, with resistance to frontline therapies common.
Consequently there is a need to understand the molecular mechanisms underlying the evasion of
cell cycle control in these cells and how this is associated with the poor prognosis seen with UC.
Here we describe a novel phenotypic screening assay that can distinguish the different phases of
the cell-cycle including the sub-divisions of mitosis. This assay uses the expression of phasespecific proteins and nuclear morphology to identify cells in G0, G1, S, G2 and M phases as well as
apoptotic cells in asynchronous cultures. Here we show assay development and validation, by use
of both siRNA and pharmacological inhibitors of specific cell-cycle checkpoints, in U2OS
osteosarcoma cells. It is applicable to many cell types and we are currently using it in several UC
cell lines, including 5637 and HT-1197 cell lines. The assay will be used to identify pro-survival
kinases in UC including those with cytostatic effects. Furthermore we will develop novel proteinbinding reagents that block the identified kinases to validate them as drug targets and to aid the
identification of small molecule therapeutics.
37 | P a g e
POSTER 15
Repurposing sulfazalazine to treat cisplatin resistant bladder cancer
Drayton RM, Dudziec E, Peter S, Bertz S, Hartmann A, Catto JW, BRYANT HE.
Academic Unit of Molecular Oncology and Academic Urology Unit, University of Sheffield
Resistance to cisplatin-based chemotherapy is a major obstacle to bladder cancer treatment. We
have identified a panel of miRNAs dysregulated in cisplatin-resistant cells. miRNA-27a was found to
target the cystine/glutamate exchanger SLC7A11 and to contribute to cisplatin resistance through
modulation of GSH biosynthesis. In patients, SLC7A11 expression was inversely related to miRNA27a expression, and those tumors with high mRNA expression or high membrane staining for
SLC7A11 experienced poorer clinical outcomes. Resistant cell lines were resensitized by restoring
miRNA-27a expression or reducing SLC7A11 activity with siRNA or with sulfasalazine.
Our findings indicate that miRNA-27a negatively regulates SLC7A11 in cisplatin-resistant bladder
cancer, and shows promise as a marker for patients likely to benefit from cisplatin-based
chemotherapy. Most excitingly SLC7A11 inhibition with sulfasalazine may be a promising
therapeutic approach to the treatment of cisplatin-resistant disease.
38 | P a g e
POSTER 16
Metal Induced Carcinogenesis and Epigenetic Dysregulation of Normal Cell Phenotype
1
1
2
2
3
CARL FISHWICK , Lucinda Cowling , Mark Dickman , Tom Minshull James Catto and Jennifer
1
Southgate
1
Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York;
2
3
Department of Chemical & Biological Engineering and Department of Urology, University of
Sheffield.
South Yorkshire has elevated rates of high grade bladder cancer, potentially linked to occupational
exposure to heavy metal species, including cadmium. Heavy metals replace normal biologically
utilized elements such as zinc and iron, resulting in disruption of enzymatic processes including
histone modification. This project aims to understand which histone epigenetic mechanisms are
affected in normal urothelial cells following cadmium exposure.
In cultured normal human urothelial (NHU) cells, exposure to non-toxic cadmium doses (10 µM)
inhibited expression of tumour-suppressor genes p16 and RASSF1A, and diminished induction of
urothelium-specific differentiation markers, including UPK2. Histone deacetylase inhibitors
recovered expression of some differentiation markers, but had no effect on others. Massspectrometric analysis of Histone H3 showed that cadmium exposure resulted in accumulation of
dimethylation of lysine 9. These observations point to candidate epigenetic-modifying enzymes,
disruption of which by cadmium could promote the observed carcinogenic events such as
attenuated expression of differentiation and tumour suppressors.
39 | P a g e
POSTER 17
The role of BMI-1 over-expression in urothelial carcinoma (UC)
1
2
3
1
Lia E. De Faveri , Jo-An Roulson , Marta Sanchez-Carbayo , Margaret A. Knowles , EMMA J.
1
CHAPMAN .
1
2
Leeds Institute of Cancer and Pathology , Department of Pathology and Tumor Biology , St James’
1
University Hospital, Beckett Street Leeds, LS97TF , U.K,
3
CIC bioGUNE, Parque Technologico De
Bizakaia, 48160, Derio Bizkaia, Spain.
Introduction
Worldwide, 380,000 new cases of UC occur annually. Expression of telomerase may be one of the
earliest steps in tumorigenesis. BMI-1 is overexpressed in telomerase-immortalised-normal-humanurothelial-cells (TERT-NHUC) compared to isogenic NHUC. The phenotypic role of BMI-1 upregulation in UC has not been fully investigated and may yield novel targets for treatment or
prevention of recurrence.
Materials and Methods
Immunohistochemistry for BMI-1 and p16 expression was performed on 75 UC and a tissue
microarray (n=92). Replication-deficient retroviral vectors were used to stably down-regulate BMI-1
expression in UC cell lines or ectopically over-express BMI-1 in NHUC in vitro.
Results and Discussion
BMI-1 was up-regulated in most UC and did not correlate with p16 expression. The role of BMI-1 in
UC is not clear although a role in mediating “stemness properties” of cancer stem cell like
populations is proposed. Ectopic overexpression of BMI-1 immortalized NHUC. NHUC-BMI-1
showed telomerase activity, bypass of senescence, reduced contact inhibition and adhesion and
increased packing density. Expression-array analysis of NHUC-BMI-1 identified an overlap with
expression profile of TERT-NHUC indicating that the role of BMI-1 in tumorigenesis may in part be
attributable to induction of telomerase activity. Knockdown of BMI-1 reduced low density colony
formation in 97-7, UC cell line.
Conclusion
Our data provides support for the notion of targeting BMI-1 as a potential therapeutic strategy.
40 | P a g e
POSTER 18
MT-MMP EXPRESSION IN HEAD AND NECK CANCER FOR PRODRUG DEVELOPMENT
R. T. ANKRAH, S. D. Shnyder, R. A. Falconer and P. M. Loadman.
Institute of Cancer Therapeutics, University of Bradford, BD7 1DB.
Head and Neck Squamous Cell Carcinomas (HNSCC), a group of highly aggressive heterogeneous
epithelial malignancies, are considered the fifth most common cancer with the seventh highest
cancer mortality. MT-MMPs are overexpressed in various tumours. In contrast, expression of active
MT-MMPs in normal tissues is largely absent. Differential gene expression of multiple MMPs in
HNSCC has been investigated, but protein expression of MT-MMPs has not.
Here, we characterise the protein expression of MT1-, MT2- and MT3-MMP in various cancers
(including HNSCC) by immunohistochemistry and Western Blot and relate these with previously
published RT-PCR data (Atkinson et al., 2007). Methods for protein identification by mass
spectrometry are currently being developed. This, in conjunction with the aforementioned
techniques will constitute a comprehensive system for characterising MT-MMP expression in
tissues. A spectrum of MT-MMP expression was seen across the different tumour types, with
interestingly HNSCC demonstrating high expression for the MT-MMPs. To be a valuable tool for
prodrug development, proteolytic activity of the expressed proteins and therefore proteolytic
capacity of tumours must be demonstrated, and this is currently being addressed.
41 | P a g e
POSTER 19
The effect of soluble factors derived from the head and neck tumour microenvironment on
the proliferation of isolated immune cell populations
JOANNE D SMITH, Nicholas D Stafford, John Greenman, Victoria L Green. Daisy Research
Laboratories, Castle Hill Hospital, School of Biological, Biomedical and Environmental Sciences,
University of Hull, HU16 5JQ.
Malignant epithelium and associated stromal cells secrete soluble factors which may influence
tumour evasion of host immunity. The effect of these factors on the proliferation of individual
immune cell populations has been investigated.
The conditioned medium (CM) from HNSCC cell lines and primary tumour-derived fibroblasts (2
oropharyngeal and 2 laryngeal) was collected after 3 days of incubation in normoxic and hypoxic
+
+
lo
conditions. The CM was added to T regulatory cells (CD4 CD25 CD127 ), T effector cells
+
-
+
(CD4 CD25 ) and cytotoxic T cells (CD8 ) isolated from healthy donors (n=4), for 48 hours before
using an MTS proliferation assay.
A significant increase in the proliferation of T regulatory cells was seen in 1 of 4 PBMC samples
after adding all CM and 1 of 4 PBMC following addition of oropharyngeal cell line CM and laryngeal
fibroblast CM (hypoxic and normoxic). A significant increase in proliferation of T effector cells was
only seen with a few CM: with 3 of 4 samples reacting to at least one CM. No conclusive effect was
+
observed on CD8 cells.
In conclusion, the secretome of HNSCC epithelial cells and fibroblasts has the ability to alter the
growth of individual sets of immune cells but this varies between samples.
42 | P a g e
POSTER 20
Tumour activated licensed NK cells produce TNFSF14, a mediator of dendritic cell
maturation and enhanced anti-tumour immunity.
1
1
1
1
2
2
Tim D. Holmes , Emma V.I. Black , Andrew V. Benest , Erica B. Wilson , Candida Vaz , Betty Tan ,
2
1
Vivek M. Tanavde and GRAHAM P. COOK .
1. Leeds Institute of Cancer and Pathology, University of Leeds School of Medicine
2. Bioinformatics Institute, A*Star, Singapore.
Natural killer (NK) cells have potent anti-tumour activity. As well as direct cytotoxic activity, NK cells
produce cytokines that modulate cellular immunity. We show that tumour-stimulated NK cells rapidly
induce the production of the TNF superfamily cytokine TNFSF14 (also known as LIGHT) and that
this cytokine allows NK cells to promote dendritic cell maturation, a key step in the initiation of T cell
mediated immunity. TNFSF14 production is restricted to a population of NK cells known as the
licensed or educated population. The licensing mechanism is a safeguard to protect healthy tissues
against the cytotoxic and inflammatory action of NK cells but can restrict NK cell activity against
tumours. These results highlight the importance of recruiting the appropriate immune subsets in
immunotherapy and identify key challenges for manipulating the immune system for more efficient
cancer immunotherapy.
43 | P a g e
POSTER 21
High risk Myelodysplastic Syndromes (MDS) patients show increased bone loss compared
to those with low risk disease
1
2
2
2
O. Gallagher , D. Bowen , C. Cargo , A. Jack and I BELLANTUONO
1
1
Department of Human Metabolism, Mellanby Centre, The Medical School, University of Sheffield;
2
Institute of Oncology, St James Hospital, Leeds
MDS is a preleukaemic disease typically in older people with limited curative therapeutic options.
The causes of the disease are unknown. Recently alteration in the bone cellular composition has
been shown to recapitulate MDS-like disease in a murine model. However, it is unknown whether
MDS patients develop the same bone alterations seen in mice. In this study we aimed at assessing
whether already archived human biopsy from MDS patients were of sufficient quality to assess bone
structure, function and cellular composition. Patients affected by Refractory Cytopenia with
Multilineage Dysplasia (low risk MDS, n=4 age range 67-77y) and Refractory Anemia with Excess
Blasts 1 (high risk MDS, n=4, age range 74-82y) were selected. 3D analysis of trephine biopsy by
microCT showed changes in bone microstructure with significant loss in trabecular bone volume
density in high risk MDS compared to low risk (8.0±3.2% vs 20±4.2%, p=0.03), decreased
trabecular number (0.9±0.3 vs 2.2±0.5 Tb N/mm p=0.03) but not thickness and increased trabecular
separation. The quality of the biopsies was sufficient for histomorphometric analysis and only 1 of
the 8 samples was discarded. Although it was possible to detect osteoclasts, osteoblasts and
osteoid, their presence was limited and variable among samples. Power calculations suggest that a
sample size approximately 100 patients per group is required to assess those parameters.
44 | P a g e
POSTER 22
Ibrutinib Inhibits B Cell Receptor Signalling-Dependent Histone Phosphorylation in Chronic
Lymphocytic Leukaemia
1
1,2
2
1,2
1
SARAH KREUZ , Talha Munir , Andy C. Rawstron , Peter Hillmen , Reuben M. Tooze , Pascal F.
Lefevre
1
1
2
Leeds Institute of Cancer and Pathology, Section of Experimental Haematology; St. James's
Institute of Oncology, University of Leeds, Leeds LS97TF
Signalling via the B cell receptor (BcR) pathway plays a major role in the pathogenesis of chronic
lymphocytic leukaemia (CLL) through alterations in gene expression, which are the direct
consequences of signal-dependent chromatin changes. However, little is known about the impact of
BcR signalling on chromatin structure. In this study, we show that cross-linking of the BcR on CLL
cells results in transcriptional activation of the downstream targets EGR1 and DUSP2 and the
concomitant phosphorylation of histone H3 threonine 6 and 11 at these genes. Moreover, we find
that Ibrutinib, which inhibits Bruton’s tyrosine kinase downstream of the BcR and is currently trialled
for CLL treatment, completely abrogates the BcR-induced changes in gene expression and histone
phosphorylation. Ibrutinib also affects methylation and acetylation of histones and RNA
polymerase II occupancy at target genes. Inhibitors of several BTK-downstream kinases can
partially mimic these effects, making those kinases potential alternative therapeutic targets for
Ibrutinib-resistant patients.
45 | P a g e
POSTER 23
Texture and Regression Tree Analysis in the Characterisation of Ovarian Lesions
Authors: PETER GIBBS, Martine Dujardin and Lindsay Turnbull
Address: MRI Centre, HYMS at University of Hull, Hull, East Yorkshire
Purpose: The presence of solid components in both benign and malignant lesions causes
diagnostic difficulty in MRI of complex ovarian masses. Correct diagnosis is important since
malignant lesions require hysterectomy, bilateral oophorectomy, omentectomy and possibly
appendectomy. This study aims to explore the utility of texture analysis in the diagnosis of ovarian
malignancy.
Methods: Data from 96 women with histopathologically proven ovarian cancer (n=67), borderline
ovarian tumour (n=28), cystadenoma (n=14) or cystadenofibroma (n=19) was retrospectively
analysed. Texture analysis was performed on T 2 weighted images using the gray level cooccurrence matrix method. Due to the uneven group sizes, which can result in over emphasis on
trying to correctly predict the largest group, repeated testing of equal sample sizes (n=14 for each
group) via random sampling was employed.
Results: Significant differences between the four groups were consistently noted for 8 parameters.
After correlation analysis and regression tree analysis using the CART algorithm a final
classification tree contained 3 parameters (f2 – contrast, f15 – cluster shade, f16 – cluster
prominence). This model correctly classifies 11.4/14 (81%) of cystadenofibromas, 9.8/14 (70%) of
cystadenomas, 8.7/14 (62%) of borderline ovarian tumours and 9.4/14 (67%) of ovarian cancers.
Conclusions: Texture analysis has been successfully applied in the diagnosis of ovarian
malignancy. After performing repeated testing a robust diagnostic model has been developed with
an overall accuracy of 70%. Appropriate use of correlation analysis and tree pruning results in a
model with only 3 parameters, thus avoiding over parameterisation.
46 | P a g e
POSTER 24
Genetic Engineering of Ovarian Cancer Cells to Identify Resistance Mechanisms to AntiVascular Agents
1
Valluru MK, Rahman A, Lyons SK , Kanthou C, Tozer GM and ENGLISH WR.
CR-UK Tumour Microcirculation Group, CR-UK/YCR Sheffield Cancer Research Centre, The
1
Medical School, University of Sheffield, S10 2RX, UK. CR-UK Cambridge Research Institute, The
Li Ka Shing Building, Cambridge CB2 2RX, UK.
Clinical trials of bevacizumab, a monoclonal antibody targeting VEGFA, have shown promise in
patients with ovarian cancer. Biomarkers are now needed to stratify patients for treatment allowing
regulatory approval to be sought. VEGFA is expressed as several isoforms and levels of VEGFA121 in particular may predict response to therapy.
The aim of this pump prime project was to generate novel ovarian cancer cell lines to explore the
link between VEGFA isoform expression and resistance to Bevacizumab. Zinc Finger Nucleases
were used to generate COV362 lines heterozygous for the wild type and a spliced allele expressing
a single VEGFA isoform (vegfa-wt/vegfa-121 and vegfa-wt/vegfa-189). Initial characterisation has
shown proliferation of wt/121 cells is inhibited by bevacizumab, whereas the proliferation of wt/wt
and wt/189 cells was not. Our poster will explore differences between wt/121, wt/wt and wt/189 cells
in vitro and in vivo.
47 | P a g e
POSTER 25
Texture analysis of 3T high resolution T2 weighted images in ovarian cystadenoma versus
borderline tumour.
1
1
1
M.I. Dujardin , P. GIBBS , L.W. Turnbull .
1
Centre for MR Investigations, University of Hull in association with Hull York Medical School, HRI,
Anlaby Road, Hull, UK.
Purpose
Preoperative diagnosis of borderline ovarian
tumour (BOT) is challenging. This study
investigates the ability of texture analysis
applied to high resolution T2 weighted imaging
(T2WI) to distinguish ovarian cystadenoma from
BOT.
Methods
Preoperative 3T T2WI
(TR=3431ms/TE=111ms/slicethickness=4mm/gap=1mm/matrix=512*416) in
cystadenoma (N=14) and BOT (N=28) were
retrospectively studied. Single slice ROIs were
drawn and texture descriptors f1-f16 were
output using in-house developed software
(Gibbs et al 2003[2]).
Results
Figure1 illustrates T2WI. A significant difference (p=0.02) was found for f14 (maximal correlation
coefficient) between both groups. Box plot results for f14 are given (Figure2). No significant
difference was observed in any of the other 15 parameters. ROC-analysis for f14 results in
AUC=0.7.
Conclusion
Texture analysis of 3T T2WI did
allow discrimination between
cystadenoma and BOT using f14,
leading to a diagnostic accuracy of
0.7. Further research is necessary
to see whether the addition of
textural information to anatomical
MR data improves preoperative
differentiation.
References
[1]Morotti et al.(2012)
ArchGynecolObstet 285;1103-1112
[2]Gibbs et al.(2003) MRM50:92-98
48 | P a g e
POSTER 26
Nac-1, a promising target for novel ovarian cancer therapeutics
MARK A. STEAD and Stephanie C. Wright
School of Biology, University of Leeds, Leeds, LS2 9JT
Ovarian cancer is the most lethal gynaecological malignancy. Current first-line chemotherapies
often result in remission; however, nearly all patients relapse and long-term survival rates are poor.
Ovarian cancers frequently exhibit increased levels of the Nac1 protein, especially in recurrent
disease, and Nac1 expression in these cancers correlates with shorter disease-free survival.
Artificial knockdown of Nac1 in cancer cell lines has been shown to induce apoptosis and rescue
the sensitivity of these cells to paclitaxel. Thus, Nac1 shows promise as a potential therapeutic
target for the treatment of these cancers.
Nac1 is a BTB-transcriptional repressor and a component of the ubiquitination pathway; BTB
domains mediate protein interactions that can result in oligomerisation and heteromeric interactions.
We have identified an interaction between the Nac1 and Miz1 BTB domains using yeast two-hybrid
assays and mammalian Co-IPs. We demonstrate that the Nac1 BTB domain is necessary for the
interaction between Nac1 and Miz1 and determined the stoichiometry of this interaction.
Furthermore, we have described the effect of Nac1 silencing on Miz1 target gene expression using
siRNA.
49 | P a g e
POSTER 27
Identification of regulatory DNA variants in apoptosis genes associated with breast cancer
risk
1
1
2
SH Rigas , I.W. Brock , NJ Camp , A COX
1
1
2
Department of Oncology, University of Sheffield, Sheffield, UK, University of Utah, Salt Lake City,
USA
Genome-wide association studies have identified over 70 low penetrance DNA variants associated
with breast cancer risk. These are mainly located in non-coding regions of the genome, and are
likely to be affecting gene expression. Our aim is to identify alleles affecting the expression of breast
cancer-related apoptosis genes (expression quantitative trait loci, eQTLs).
Polymorphic variants in the CASP8 gene region were genotyped on blood or normal breast tissue
DNA from 88 women with breast cancer attending the Huntsman Cancer Centre in Salt Lake City.
Gene expression was measured in normal breast tissue for 96 apoptosis-related genes by use of a
qRT-PCR Apoptosis Array (Life Technologies) and by RNA-seq.
Variants associated with increased risk of breast cancer were associated with reduced expression
of CASP8 mRNA (p<10-3) and variants associated with a lower risk of breast cancer were
associated with reduced levels of CASP10 and CFLAR mRNA. These results suggest that variants
in the CASP8 region on chromosome 2q may affect breast cancer risk through both apoptosis and
autophagy pathways. Other reports suggest that autophagy inhibition might be a potential
therapeutic strategy for triple negative breast cancers, which currently lack an effective targeted
treatment.
50 | P a g e
POSTER 28
Molecules and mechanisms central to breast cancer metastasis to bone: A quantitative
proteomics approach
1
2
3
1
5
JULES A WESTBROOK , David A Cairns , Caroline A Evans , Penny Ottewell , David N Perkins ,
6
3
7
7
1
Carl Smythe , Phillip C Wright , Valerie Speirs , Andrew M Hanby , Ingunn Holen , Robert E
1
1
Coleman , Janet E Brown
1
Academic Unit of Clinical Oncology, Weston Park Hospital, University of Sheffield
2
Clinical Trials Research Unit, Leeds Institute of Clinical Trials Research, University of Leeds
3
Department of Chemical and Biological Engineering, University of Sheffield
5
Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Australia
6
Department of Biomedical Science, University of Sheffield
7
Leeds Institute of Cancer and Pathology, University of Leeds, St James's University Hospital,
Leeds
Despite major progress in breast cancer management, many patients still progress to advanced,
incurable disease. The majority develop bone metastases and the consequent severe bone pain
and other skeletal complications which substantially limit quality of life. This project builds on a
successful, SILAC proteomics-based, YCR pump-priming award which demonstrated a substantial
change in protein expression profile when MDA-MB-231 human breast cancer cells developed
bone-homing capacity after in vivo passages in nude mice. This has been shown to be a good
model for human bone metastasis. Using stable cells from seven such passages representing
different cell phenotypes as bone-homing capacity develops, we are carrying out a study of these
cells using proteomic approaches (spike-in SILAC and phosphoproteomics) to further define the
pathways and molecules that play a key role as breast cancer cells develop capacity to disseminate
to and grow in bone, and to identify potential protein biomarkers to predict the risk of development
of bone metastases in breast cancer patients. Initial validation will be performed in patient tissue
and blood, using tissue banks specially collected for this purpose in the large AZURE study. We will
present the initial progress in this project which commenced in January 2014.
51 | P a g e
POSTER 29
Associations between survival intervals and MR imaging in breast cancer patients
undergoing neoadjuvant chemotherapy: a comparison with traditional prognostic indicators
1
1
2
Dr MARTIN D PICKLES , Dr Martin Lowry , Dr David J Manton , Professor Lindsay W Turnbull
1
1
Centre for MR Investigations, HYMS at University of Hull, HRI, Anlaby Road, Hull
2
Radiation Physics Department, Hull & East Yorkshire Hospitals NHS Trust, CHH, Hull
Objectives: Examine associations between dynamic contrast enhanced MR imaging (DCE-MRI)
and survival intervals, in a cohort with locally advanced breast cancer treated with neoadjuvant
chemotherapy (NAC), surgery and adjuvant therapies. Further, to compare the prognostic value of
DCE-MRI parameters against traditional survival indicators.
Methods: DCE-MRI parameters were obtained prior to treatment and post 2
nd
NAC cycle. Biopsy
samples and clinical examination provided traditional pre-treatment survival indicators. To
demonstrate which parameters were associated with disease free (DFS) and overall survival (OS),
Cox’s proportional hazards models (CPHM) were employed.
Results: When considering DFS positive axillary nodal status (hazard ratio [HR] 6.79), younger age
(HR 3.37), negative oestrogen receptor status (HR 3.24), pre-treatment MaxEI (HR 6.51) and
percentage change in MaxEI between baseline and 2
nd
cycle NAC (HR 1.02) represented the retained
CPHM covariates. Similarly, positive axillary nodal status (HR 11.47), negative progesterone receptor
status (HR 4.37) and percentage change in AUC90 following 2
nd
cycle NAC (HR 1.01) represented the
retained predictive CPHM variables for OS.
Conclusion: DCE-MRI parameters obtained prior to NAC and/or post 2
nd
cycle can provide
independent prognostic information that can complement traditional prognostic indicators available
prior to treatment.
52 | P a g e
POSTER 30
Circulating cell-free plasma DNA: optimising extraction to develop a biomarker in lung
cancer
Authors: F TAYLOR
1+2
2
3
, P J Woll , M D Teare , A Cox
1
1) Academic Unit of Molecular Oncology, The Medical School, University of Sheffield,
Sheffield, S10 2RX
2) Academic Unit of Oncology, CR-UK/YCR Sheffield Cancer Research Centre, Weston Park
Hospital, Whitham Road, Sheffield, S10 2SJ
3) School of Health and Related Research (ScHARR), University of Sheffield, S10 2RX
Tumour mutations are identified in circulating cell-free DNA (cfDNA) in the blood of lung cancer
patients. CfDNA is therefore a source of potential biomarkers to aid early detection. However, the
quantities of cfDNA in the blood are measured in ng/ml. This study aims to maximise DNA yield to
enable sensitive downstream mutation analysis.
DNA was extracted from healthy volunteer plasma samples spiked with tumour DNA and matched
un-spiked samples and the percentage of DNA recovery was calculated. Extracted DNA was
quantified by SYBR green qPCR. Whole genome amplification was followed by PCR of the BRAF
V600E region.
There was no significant difference in the mean percentage of DNA recovered between the QIAamp
Blood Mini Kit and phenol chloroform method (6.3 % vs 8.3 % t-test p=0.37, N=3). Extracted DNA
was amplified the greatest by the GenomePlex WGA2 kit compared to the Illustra GenomiPhi V2 kit
(N=1). We intend to extract plasma DNA from lung cancer cases and controls in the ResoLUCENT
study with the QIAamp Blood Mini Kit because it is quick and simple to use. Next generation
sequencing with the Ion Torrent platform to detect lung cancer mutations will be used initially to
assess cfDNA as a potential biomarker.
53 | P a g e
POSTER 31
Downstream consequences of cancer-associated CIZ1 variant expression
DORIAN R.A. SWARTS, Gillian Higgins, Heather Sercombe, Christopher Smith, Dawn Coverley
Department of Biology, University of York, YO10 5DD
CIZ1 is an oestrogen-responsive gene that promotes tumour formation in mice. Alternatively spliced
variants are linked with medulloblastoma and Ewing’s Tumour, and with lung cancer where variant
expression is being developed as a blood test for early stage disease. CIZ1 functions to regulate
DNA replication in relation to the nuclear matrix (NM), however, the impact of alternatively spliced
variants on cellular function is not understood.
We show here that exposure to oestrogen promotes alternative CIZ1 splicing in breast cancer cells,
to produce a signature that is evident in common solid tumours of breast, colon, lung and other
tumour types. Our goal is to identify the exact variants that are responsive to oestrogen and the
consequences of their expression for (cancer) cells.
We have validated specific detection tools for all 17 CIZ1 exons and profiled individual changes in
expression in response to oestrogen. Using quantitative RT-PCR we measured a ~2-fold (P=0.015)
overrepresentation of the C-terminal NM-attachment domain but not the N-terminal replication
domain, in response to oestrogen in MCF-7 breast cancer cells. At 1 hour after induction, this
response preceded the main expression spike of common oestrogen-responsive genes (MYC and
STC2), consistent with a role in reprogramming gene expression.
54 | P a g e
POSTER 32
Development of an analytical assay to assess polysialyltransferase inhibition
SMI ElKASHEF , M SUTHERLAND, SD SHNYDER, LH PATTERSON, PM LOADMAN and RA
FALCONER
Institute of Cancer Therapeutics, School of Life Sciences, University of Bradford, Bradford BD7
1DP, U.K.
Polysialic acid is a homopolymer of α-2,8-linked sialic acid residues which is added to specific Nglycans on NCAM. Polysialylation is a unique posttranslational modification of NCAM and is
regulated by two polysialyltransferase enzymes. Polysialic acid is over-expressed during the
progression of a number of malignant human tumours. In these tumours, polysialylation of NCAM
correlates with increased metastatic potential and poor prognosis. Our hypothesis is that inhibition
of polysialyltransferase enzymes will inhibit tumour metastasis and consequently improve
prognosis.
Here we describe the development of an analytical technique to assay small molecule
polysialyltransferase inhibitors, using a fluorescent acceptor (DMB-DP3). DMB-DP3 was
synthesised and purified in a single step from commercially available starting materials. Two
purification strategies were then developed using RP-HPLC and anion exchange-HPLC. The
polysialylation reaction was then studied using CMP as a prototype inhibitor. A concentrationdependent reduction in DMB-DP4-Sia (the product of the polysialylation of DMB-DP3-Sia) was
observed and quantified, thereby validating the assay.
HPLC separation coupled with fluorescence detection of reaction products enabled precise and
quantitative detection of polysialyltransferase activity. To-date this is the only analytical technique
that provides a simple and high throughput, analysis of human polysialyltransferase inhibitors.
55 | P a g e
POSTER 33
CDK18, A novel human Cyclin-Dependent Kinase required for efficient DNA replication and
genome stability.
Giancarlo Barone1, Christopher S. Staples1, Katie N. Myers1, Karl W. Paterson1, Edward E.
McKenzie2, Claire E. Eyers3, Patrick A. Eyers3 & Spencer J. Collis1
1Accademic Unit of Molecular Oncology, CR-UK/YCR Sheffield Cancer Research Centre,
Department of Oncology, University of Sheffield.
2Protein expression facility, Manchester Institute of Biotechnology, University of Manchester.
3Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK
Genetic instability is a hallmark of cancer, and the DNA Damage Response (DDR) in coordination
with cell cycle checkpoints monitors DNA damaging events to supress genome instability. As such,
genetic disruption of these pathways can lead to cancer predisposing disorders; however, targeting
these factors in cancer cells can be exploited to improve therapeutic responses to both radiotherapy
and chemotherapeutic regimes. From a human genome-wide RNAi screen for novel DDR factors,
we have identified the previously uncharacterised Cyclin-Dependent Kinase 18 (CDK18) as a novel
regulator of genomic maintenance. Cells deficient in this kinase exhibit chromosomal alignment
defects and increased genomic instability as measured by elevated levels of DNA damage. In
addition, CDK18 depletion leads to temporal effects on S-phase progression and inefficient DNA
replication. These cells are further characterised by an increased number of Cajal Bodies, which are
associated with defective early DNA replication events. Taken together, we have identified a novel
CDK, which upon depletion causes increased genome instability brought about by dysregulated
DNA replication events leading to chromosomal aberrations.
56 | P a g e
POSTER 34
The microRNA biogenesis machinery is inherently connected with endothelial cell survival
and tumour-associated angiogenesis
Matthew Warner
Dimitris Lagos
1,2,3
1,3
1,3
1,2
, Bailey Massa , Maria Chatzifrangeskou , Jessica Haslam ,
1,2,3
1. Centre for Immunology and Infection, University of York, York, U.K.
2. Department of Biology, University of York, York, U.K.
3. Hull York Medical School, University of York, York, U.K.
The ANG/TIE-2 system is essential for embryonic vasculature development, and postnatal
angiogenesis and deregulation of this signaling axis is invariably associated with tumour-associated
angiogenesis and lymphangiogenesis. Despite microRNAs (miRNAs) being shown to play a critical
role in the regulation of the vasculature, it remains unclear how the ANG-TIE2 axis regulates miRNA
expression in human endothelial cells. Intriguingly, our studies reveal that the ANG/TIE-2 axis
regulates human dermal lymphatic endothelial cell (HDLEC) miRNA biogenesis by regulating the
phosphorylation and expression of TRBP, a critical component of the cytoplasmic miRNAprocessing protein assembly. Overall, our findings reveal an inherent connection between the TIE2
receptor tyrosine kinase, critical signaling pathways and control of miRNA cellular outputs, thus
having significant implications for the development of anti-angiogenic cancer therapies.
57 | P a g e
POSTER 35
Small molecule tetraazamacrocyclic CXCR4 chemokine receptor antagonists: applications in
imaging and therapy
B.P.BURKE, R.Smith, K.L.Nicholson, C.Cawthorne and S.J.Archibald*
Department of Chemistry and Positron Emission Tomography Research Centre, University of Hull,
Cottingham Road, Hull, HU6 7RX.
The chemokine receptor CXCR4 plays a key role in both normal and pathological physiology and
has been shown to be overexpressed on over 23 cancer types. Recently, many groups including
ourselves have been developing small molecule antagonists to investigate their therapeutic
potential in cancer treatment. In the main, these molecules are either arginine and lysine rich
peptides or bis-tetraazamacrocycles which target aspartate residues on the extracellular protein
surface.
Here, we present the development of our novel bis-tetraazamacrocyclic CXCR4 antagonists in
which configurational restriction and metal complex formation effects a dramatic increase in both
binding affinity and receptor residence time. We are also investigating the advantages of this
approach in Positron Emission Tomography (PET) and Single-Photon Emission Computed
Tomography (SPECT) imaging for improved cancer diagnosis, patient stratification and monitoring
of treatment progression.
58 | P a g e
POSTER 36
Potential of novel chemokine antagonists for the treatment of cancer
M.V. Vinader, M. S. Ahmed, H. A. D. Basheer, D. Ahmet, S. D. Shnyder, L. H. Patterson, K. Afarinkia
Institute of Cancer Therapeutics, University of Bradford, BD7 1DP, United Kingdom
Chemokines, particularly CXCL12, acting on CXCR4 receptor, and CCL19 and CCL21, acting on
CCR7 receptor, have a multifaceted involvement in the regulation of various tumour cell functions,
including growth, angiogenesis, survival, migration and immune evasion, and in particular, play a
critical role in the homing of tumour cells into metastatic target organs. Chemokines CCL19 and
CCL21 are abundant in the lymph nodes and control the growth and lymph node metastasis in
cancers that express CCR7, including breast, oral squamous cell and colon. CXCR4 is the most
widely expressed chemokine receptor in tumours and promotes organ specific metastasis in many
types of cancer. We describe the discovery and present preclinical data for ICT5122, a novel
CXCR4 antagonist and ICT5888, a novel CCR7 antagonist.
59 | P a g e
POSTER 37
Development of Novel Dual/Multi-Integrin Antagonists as Improved Anticancer Agents
ADAM THROUP, Andrew Gordon, Fatemah Al-Shammari Hanadi Ahmedah, Laurence Patterson,
Steven D Shnyder, Mark Sutherland, Helen M Sheldrake
Institute of Cancer Therapeutics, University of Bradford
Introduction:
Integrins are a family of cell surface receptors that mediate cell-ECM and cell-cell adhesion
along with transmembrane signalling. They are involved in cell migration, proliferation,
differentiation, angiogenesis and apoptosis.
The RGD binding integrins are often upregulated in cancers notably melanoma, prostate cancer and
glioblastoma. Their ability to increase survival, proliferation, metastasis, invasion and angiogenisis,
makes antagonism of these receptors an interesting therapeutic target.
Methods and Results:
A library of >100 RGD mimetics have been designed and synthesised, based upon molecular
modelling of known integrin ligands and integrin X-ray crystal structures.
Appropriate cell lines and assays for testing have been identified and the compounds were
screened for αvβ3 antagonism in adhesion, migration and invasion assays and αIIbβ3 antagonism
in a platelet aggregation assay showing a range of activities.
Conclusion:
We have identified a number of novel highly active β3 integrin antagonists for further
development.
The RGD-mimetic skeleton has shown potential for modification into a multi-integrin antagonist.
60 | P a g e
POSTER 38
Novel Pt based photodynamic chemotherapeutics
Luke McKenzie, Rachel E. Doherty, Igor Savanovic, Sarah Bottomley, Julia Weinstein and Helen E.
Bryant
We have previously reported a platinum-based compound which absorbs strongly at 405 nm light
and which is readily taken up by cells, predominantly residing in the nuclear compartment. The
present study demonstrates this compound has high cytotoxicity in the nM dosage range following
exposure to 405 nm light whilst remaining non-toxic to cells in the absence of light. Furthermore,
light-activated PtNCNMe-induced cell death in cisplatin-resistant cancer cells was observed,
highlighting it as a potential therapeutic alternative to conventional Pt based chemotherapies. We
provide evidence that the mechanism of action of light-activated PtNCNMe-induced cell death is
likely due to irreversible DNA damage through PtNCNMe intercalation with DNA and subsequent
generation of ROS which in turn induces SSBs in DNA. Progression of this work will involve
activation of PtNCNMe at a longer wavelength of light, near IR light, in order to penetrate deeper
into tissue and take advantage of its PDT effect in a wide range of tissues.
61 | P a g e
POSTER 39
Novel Sydnone-Based Vascular Disrupting Agents for Use in Cancer Therapy
a,b
b
b
a
ANDREW W. BROWN , Gillian M. Tozer , Chryso Kanthou and Joseph P. A. Harrity
a
Department of Chemistry, Dainton Building, Brook Hill, University of Sheffield, S3 7HF
b
The Medical School, University of Sheffield, Beech Hill Road, S10 2RX
Combretastatin-A4-phosphate (CA-4-P) is a tubulin-binding agent that causes rapid collapse of the
vascular network of tumours. CA-4-P is currently undergoing clinical trials as a potential anticancer
therapeutic, but suffers from solubility and stability issues. The aim of this study was to synthesise
more stable combretastatin analogues containing a sydnone motif as the scaffold and screen
activity using endothelial cell models.
A variety of sydnones were synthesised from the corresponding amino acid via N-nitrosation,
followed by cyclodehydration with trifluoroacetic anhydride. Novel, palladium-mediated direct
arylation of sydnones with aryl chlorides generated analogues in good to excellent yields.
Analogues were used to treat cultured human umbilical vein endothelial cells and their effects on
the endothelial cytoskeleton were studied by immunofluroescence. Effects on proliferation were
studied by culturing the cells for 3 days in the presence of the drugs followed by staining with crystal
violet.
25 analogues have been successfully synthesised. The most active compound inhibited endothelial
proliferation with an IC50 of 24 nM. This analogue also caused microtubule disruption and actin
stress fibre formation at doses of ≥0.5 µM.
Sydnone analogues displayed activity comparable to CA-4-P against endothelial cells. Structure
activity relationships (SARs) are being utilised to design more effective drugs.
62 | P a g e
POSTER 40
Exploiting cancer cell metabolism to potentiate DNA-damaging anti-cancer agents
selectively in cancer cells
1
2
SIMON J ALLISON , Filippo Minutolo and Roger M Phillips
1
1
Institute of Cancer Therapeutics, University of Bradford, UK
2
Dipartimento di Farmacia, Università di Pisa, Italy
Metabolic differences between cancer and non-cancer cells present opportunities for more selective
anti-cancer therapeutic targeting. Many cancer cells rely upon aerobic glycolysis (the Warburg
+
effect) to fuel their growth. The enzyme LDH-A is key to regenerating the co-factor NAD that is
required for aerobic glycolysis. We show that suppressing LDH-A, either by RNAi or a small
molecule inhibitor, is able to potentiate the activity of DNA-damaging anti-cancer drugs selectively in
cancer cells. LDH-A suppression reduced the IC50 of the topoisomerase I inhibitor camptothecin by
~2-fold in HCT116 cancer cells, correlating with elevated levels of double-strand DNA breaks and
significantly increased cancer cell death. Similar potentiation was observed for the topoisomerase II
inhibitor etoposide and LDH-A suppression significantly increased γH2AX phosphorylation induced
by the pro-drug EO9 and the methylating agent temozolomide. As the DNA repair enzyme PARP
+
requires NAD for its activity, we are investigating whether cancer-selective potentiation of DNA+
damaging anti-cancer agents by LDH-A suppression is due to effects on PARP activity via NAD .
63 | P a g e
POSTER 41
Targeting protein ubiquitination pathways in cancer therapy
Mark A. Stead, Laura K. van Geersdaele, and Stephanie C. Wright
School of Biology, University of Leeds, Leeds, LS2 9JT
Ubiquitination is a post-translational modification that targets proteins for degradation by the 26S
proteasome. The regulated ubiquitination of specific proteins is critical for fundamental biological
processes, and alterations in ubiquitination pathways are implicated in many human cancers.
The E3 ubiquitin-protein ligases interact with substrates to carry out the final stage of the
ubiquitination reaction, and various adaptor proteins act to recruit specific substrates for
ubiquitination. SPOP is an adaptor protein of the cullin3 E3 ligase complexes; it has been
implicated both as an oncogene and as a tumour suppressor in human cancer. We have shown
that SPOP forms high-order oligomers in mammalian cells, and have used X-ray crystallography to
determine the detailed structure of these complexes. We propose that the overexpression of SPOP
in tumours leads to an increase in its oligomerisation, leading to the enhanced ubiquitination and
degradation of a subset of its substrates. The crystal structures will enable us to develop inhibitors
of SPOP that selectively block its high-order oligomerisation as a therapeutic strategy.
64 | P a g e
POSTER 42
METHOTREXATE INHIBITS JAK/STAT SIGNALLING.
1
2
3
2
1,4
SALLY THOMAS , Katherine Fisher , Steve Brown , Kirsty Johnstone , Sarah Danson , John
1,5
1,2
Snowden , Martin Zeidler .
1. Sheffield Cancer Research Centre. 2. Department of Biomedical Sciences, University of
Sheffield. 3. Sheffield RNAi Screening Facility, University of Sheffield. 4. Weston Park Hospital,
Sheffield. 5. Royal Hallamshire Hospital, Sheffield.
The JAK/STAT signalling pathway transduces signals from cytokines controlling proliferation,
differentiation and apoptosis. JAK/STAT activation occurs in many cancers, including
myeloproliferative neoplasms which are frequently caused by a mutation (V617F) in JAK2. Inhibiting
JAK/STAT activation is an attractive therapeutic approach for these disorders.
We performed a screen of 2000 small molecules in Drosophila and identified methotrexate as
a strong inhibitor of JAK/STAT activation. In human cells methotrexate reduced phosphorylation of
specific STATs without affecting phosphorylated proteins in other signalling pathways. Methotrexate
significantly reduced STAT5 phosphorylation in cells expressing JAK2 V617F. This effect was not
reversed by folinic acid. Furthermore, methotrexate-treated cells retained the capacity to activate
the pathway when stimulated with erythropoietin. These effects occurred at drug concentrations
equivalent to those seen in patients.
Our results identify a novel mechanism of action for methotrexate and suggest that low dose
oral methotrexate may be an alternative treatment for patients with myeloproliferative neoplasms.
65 | P a g e
POSTER 43
Control of blood vessel lumen morphogenesis by DOCK4
1,2
1
3
1
1
Sabu Abraham , Margherita Scarcia , Rick Bagshaw , Kathryn McMahon , Gary Grant ,
1
1
1
1
1
Tracey Harvey , Maggie Yeo , Helene H. Thygesen , Filomena Esteeves , Pam Jones , Valerie
1
1
1
1
4
3
Speirs , Andrew M. Hanby , Peter Selby , Mihaela Lorger , Neil Dear , Tony Pawson ,
2
Christopher J. Marshall and GEORGIA MAVRIA.
1
4
Leeds Institute of Cancer and Pathology, Leeds Institute of Molecular Medicine,
Wellcome Trust Brenner Building, St James’ University Hospital, Leeds LS9 7T.
2
Institute of Cancer Research, Division of Cancer Biology, 237 Fulham Road, London SW3
6JB, UK.
3
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto,
Ontario, Canada.
Signalling to the actin cytoskeleton is central to growth factor signalling during angiogenesis.
The actin cytoskeleton is regulated through the activity of Rho GTPases that cycle between
active GTP and inactive GDP-bound states. During angiogenesis Rho GTPases are known to
influence endothelial cell migration and cell-cell adhesion, however it is not known whether they
control formation of blood vessel lumens which are essential for blood flow. In an organotypic
system that recapitulates distinct stages of VEGF-dependent angiogenesis we show that lumen
formation requires lateral cell-cell adhesions mediated through cytoskeletal remodeling and
generation of tubule lateral filopodia. Inhibition of lateral filopodia through knockdown of the
guanine nucleotide exchange factor DOCK4 or DOCK4 heterozygous genetic deletion,
compromises blood vessel lumen size in the organotypic tissue culture system and in brain
tumours in vivo, while lengthening and anastomosis proceed uninterrupted. This results in
thinner blood vessels that do not sprout. We report a novel signaling cascade downstream of
VEGF comprising Rho GTPases arranged in tandem and Rho regulators that control lateral
filopodia formation necessary for subsequent stages of lumen morphogenesis. These findings
could be used to develop new strategies to prevent angiogenesis, and the process of
metastasis through reducing tumour blood vessel size.
66 | P a g e
POSTER 44
Trojan horse delivery of an Oncolytic Virus for Treatment of Multiple Myeloma
Simon Tazzyman, JACK HARRISON, Claire Lewis, Munitta Muthana and Andrew Chantry
University of Sheffield, Medical School, Beech Hill road, Sheffield, S10 2RX
Multiple myeloma remains an essentially incurable malignancy. In the vast majority of cases,
tumour bulk may be substantially reduced by conventional and novel chemotherapies. However,
almost invariably, disease re-accumulates and patients eventually succumb. Novel strategies to
eliminate residual plasma cells are urgently needed. Recently, a novel macrophage-based, viral
therapy designed to augment conventional anti-cancer therapies has shown therapeutic efficacy
in murine models of prostate cancer. We have adapted a similar strategy in models of myeloma.
We have developed a virus whose replication is restricted to myeloma. Additionally we are testing
a herpes simplex virus for use with our myeloma cells in an effort to have a second virus for use
in the Trojan horse system. Both these viruses induce tumour cell death when compared to uninfected cells or control viruses. With the adenovirus inducing a doubling of cell death after 6days
of infection while HSV virus induces a potent cell death after just 24-48 hours of infection with
95% of cells dead.
67 | P a g e
POSTER 45
Antibotoxome™, a novel cytotoxic conjugate, shows substantial cancer cell death in in vitro
models of pancreatic, liver, breast, cervical cancer and myeloma but is not toxic to healthy cells
Guillaume Hautbergue1, Andrew Chantry2, Simon Tazzyman2, Ali Khurram3, Matt Bryan4, Maria
Fragiadaki5
1
Institute for Translational Neuroscience (SiTRan), University of Sheffield, 2Academic Unit of
Nephrology, Department of Infection and Immunity, University of Sheffield, 3Sheffield Myeloma
Research Team, Department of Oncology, University of Sheffield, 4 School of Clinical Dentistry,
University of Sheffield, 5Department of Cardiovascular Science
Antibotoxome™ is a construct comprising a magnetic bead conjugated to two cytotoxic agents
(BLF-1 and TRAIL) and an antibody to an internalising cell surface receptor (eg CXCR4).
Burkholderia lethal factor one (BLF1) is a bacterial endotoxin produced by Burkholderi
pseudoallei. BLF1 is only toxic to cells if internalised. TRAIL binds to death receptors present on
tumour cells and is internalised into the cell inducing apoptosis via caspase-8. Tumour cells
express death receptors but healthy cells express decoy receptors rendering TRAIL toxic to
tumour cells but not healthy cells. Anti CXCR4 antibody binds to CXCR4 expressed on tumour cell
surface and leads to internalization. CXCR4 is expressed on the surface of myeloma cells.
Antibotoxome™ is thus designed to be toxic to cancer cells but not to healthy tissue. We have
performed in vitro studies demonstrating that Antibotoxome™ is toxic to cancer cell lines
including pancreatic, liver, breast, cervical cancer and myeloma. Furthermore, Antibotoxome™
has not been toxic to healthy cells including HEK293 embryonic kidney cells and MDCKII kidney
tubular epithelial cells. We now seek further funding to conduct in vivo studies using a murine
model of myeloma to treat mice bearing heavy loads of myeloma cells with Antibotoxome™ to
assess tolerability and efficacy.
68 | P a g e
POSTER 46
Game Technology Against Cancer (GTAC) ft Osteolyticon
TM
Cassie Limb (artist), Mike Futcher aka YogYog (animator), Andrei Pambuccian (programmer), Andy
Chantry (musician and medic)
Sheffield Myeloma Research Team, Dept of Oncology, University of Sheffield
Objectives
To utilize the collective consciousness of game players worldwide to defeat cancer.
To design a game that would educate patients, researchers, funders and the public about cancer
induced bone destruction.
To use game technology to design new strategies against cancer.
Methods
An artist, an animator, a programmer, a musician and a medic had a series of conversations and
dreamt up Osteolyticon. Using original textures and designs and an original soundtrack, much of
which was created using unusual digitally processed vocal sounds, the visual and aural themes for
Osteolyticon were created. Using the Blender animation package, a dynamic bone marrow microenvironment was created featuring bone continually remodeling, osteoclasts repairing microfractures and osteoblasts laying down new osteoid. Malignant plasma cells emerge and recruit new
osteoclasts to accelerate bone destruction and inhibit osteoblasts. Chemotherapy destroys the
cancer cells and bone recovers but give too much and toxic death ensues. You control, you treat,
you choose!
Results
An interactive game that is fun to play, teaches you about cancer and bone destruction and allows
you to interact with all the key elements. Osteolyticon looks particularly good projected on to large
screens and plans are afoot to project Osteolyticon, at night, onto the entire facia of the Royal
Hallamshire Hospital as part of the forthcoming University of Sheffield Festival of the Mind. When
exhibited at the recent Sheffield Cancer Research Centre, 93% of participants expressed the belief
that Osteolyticon was an effective way to engage the public. Difficulties encountered included
arguments between the artist, the animator and the medic concerning the inclusion of various New
Age elements and the tendancy of the medic to lapse into unintelligible jargon.
Conclusion
Osteolyticon unleashes unpredictable elements in the battle against cancer.
69 | P a g e
POSTER 47
TM
Osteolytica , an image analysis software package that substantially improves the accuracy
of cancer-induced osteolytic lesion measurements compared to other currently available
methods
1
2
1
2
1
Holly Evans , Twin Karmakharm , Michelle Lawson and Paul Richmond Andrew Chantry
1
Sheffield Myeloma Research Team, Dept of Oncology, University of Sheffield, UK.
2
School of Automatic Control Systems Engineering, University of Sheffield, UK.
Objectives:
Osteolytic lesions are a defining characteristic of myeloma and other cancers that destroy bone.
Measurement of these lesions is notoriously difficult and results often suffer from inaccuracy, poor
reducibility and bias. We sought to improve the accuracy and reproducibility of osteolytic lesion
analysis.
Methods:
TM
We have developed Osteolytica , an osteolytic lesion analysis software package, featuring an easy
to use step-by-step interface. It utilises novel graphic cards acceleration (parallel computing) and 3D
rendering to provide rapid reconstruction and analysis of osteolytic lesions. Analysis of osteolytic
lesions in 3 different murine models of myeloma was performed using Osteolytica
TM
and 2-D image
analysis using ImageJ software. For study 1, C57Bl/KaLwRij mice were injected with PBS (n=4) or 2
6
x 10 5TGM1 cells (n=7), and sacrificed after 21 days. For study 2, NOD/SCID-GAMMA mice were
6
injected with PBS (n=4) or 1x10 JJN3 cells (n=5) and sacrificed after 21 days. For study 3,
6
NOD/SCID-GAMMA mice were injected with PBS (n=5) or 1x10 U266 cells (n=8) and sacrificed
after 8 weeks. Tibiae were scanned using microCT and datasets reconstructed.
Results:
For study 1, the disease group exhibited significantly increased osteolytic bone disease compared
TM
to controls using Osteolytica , but not when using the 2-D method. For study 2, the disease group
exhibited significantly increased osteolytic bone disease compared to controls when using
Osteolytica
TM
and the 2-D method. For study 3, the disease group exhibited no significant difference
in osteolytic bone disease compared to ontrols using either Osteolytica
TM
or the 2-D method.
We also deployed four independent researchers to analyse 5 bones using Image J 2-d analysis.
Inter-user variability was substantial (+/- 20%). This is in contrast to multiple repeat analyses using
Osteolytica
TM
which demonstrated zero variability.
Conclusions:
Osteolytica
TM
is a novel, accurate and reproducible method of analysing osteolytic lesions. It is a
substantial improvement on previous methods, being rapid and easy to use, and eliminates interuser variability.
70 | P a g e
Be a YCR Star
We wouldn’t be here for the people of Yorkshire without the generosity of our
supporters. Every year thousands of people support us in our goal to help the people
of Yorkshire avoid, survive and cope with cancer.
Join our growing team of YCR stars
 Take on a Challenge - walk, run, cycle or go extreme
with a skydive
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or colleagues
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at an event
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Here’s a taster of some of the Events taking place this year
th
8 June
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3 August
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11 August
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11 December
Hull 10K
RU Taking the P
Great Yorkshire Bike Ride
Pennine 10K
Leeds 10K
York 10K
PGA Leeds Cup Pro Am
Dales Rider
Great North Run
Knaresborough Golf day
Berlin Marathon
Great Yorkshire Run
Yorkshire Marathon
Winter Ball
For more information on these events and other ways to get involved visit
www.ycr.org.uk and the “support us “ section
71 | P a g e
Chief Executive
Mr Charles Rowett
Yorkshire Cancer Research
39 East Parade
HARROGATE
HG1 5LQ
Registered Charity No. 516898
Tel: 01423 501269
Email: [email protected]
72 | P a g e

25 June 2014 Annual Scientific Meeting Pavilions of Harrogate

Transcription

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