William's Fund

Childhood Cancer Research Fund
John Radcliffe Hospital, Oxford
(Reg. charity no. 1057295)

Read the December 2010 update from the researchers here

Chris Mitchell - Research Update April 2010 - The development of aptamer targeting as an approach to deliver nanotherapeutics to cancer cells/tumour vasculature.

Aptamers are selected nucleic acid binding species with affinities and specificities for protein targets that rival those of monoclonal antibodies. Furthermore, aptamers have definite advantages over antibodies, in that they can be chemically synthesized and modifications can be introduced that improve their stabilities and pharmacokinetic properties. To date most approaches selectively targeting nanoparticles have used antibodies or antibody derived molecules. In this project we are investigating the potential of aptamers to

(i) target specific cancer cells and

(ii) develop strategies for aptamer nanoparticle coupling, testing and separation.

Current progress and future work

Target specific cancer cells. An aptamer library was prepared containing 1018 different aptamer molecules. Optimal conditions have been determined to exponentially amplify the library and we are currently in the process of enriching for binding to the neuroblastoma line SKN-SH. Enrichment for aptamers is a lengthy process with 20-30 phases of binding of the aptamer library to the cell line followed by amplification of the bound aptamers to enrich for the high efficiency binding molecules. A relatively inexperienced 12 month sandwich placement project student is currently working on this project. Much of the period so far has been taken up with training in the various procedures required and setting up new protocols (tissue culture, PCR etc).

Future work

Next 5 months: Over the next 5 months we will identify aptamers binding neuroblastoma cells under different conditions of cell harvesting (i.e trypsin, acutase and scraping). A negative selection protocol will be developed to remove aptamers which bind non-specifically. The final aptamer panel will be tested for binding to other cancer and non-cancer cell lines to assess specificity.

Develop strategies for aptamer nanoparticle coupling, testing and separation. Cells readily take up nanoparticles giving high levels of non-specific uptake which is both a problem in the laboratory and in a clinical setting. In collaboration with groups in the Department of Engineering Science at Oxford and Professor Jeung Sang Go currently on sabbatical from Pusan National University (PNU), Korea we are developing approaches to separate nanoparticles when coupled to aptamers/antibodies

Magnetic Separation of antibody coupled nanoparticles - manufacture of the device is depending on a £7k application for funding support from the University of Oxford, John Fell Fund - click to enlarge.

We currently have a 2 year MSc student who is developing separation approaches for aptamer bound nanoparticles. The project involves the coupling of the well characterised aptamer to the cancer marker MUC1 to fluorescently labelled nanoparticles.

Progress to date: MUC1 aptamers have been coupled to FITC labelled nanoparticles. Coupled particles currently have aggregation problems which
are being addressing. pH changes and coating particles with anti-aggregation agents will be assessed.

Long term objectives

Depending upon future funding and the results of pilot experiments there are a number of areas which could be investigated

Aptamer targeting of drugs targeted to reduce cancer cell invasion. Dr Elizabeth Rapa & Dr Sophie Hill identified a number of genes involved in cell invasion. Using an aptamer targeting approach cancer cells could be targeted in the circulation with nanoparticles containing siRNA which down regulates cell invasion genes. Thus reducing cell invasion and hopefully metastatic disease. In addition to cell targeting, release of the siRNA once inside the cell and selecting the appropriate nano vector are key areas to address.

Aptamer targeting of diagnostics to monitor treatment responses in vivo. Our aim is target specific angiogenic markers associated with new tumour vasculature as a way of rapidly monitoring the effectiveness of new cancer therapies. Allowing new drugs to be assessed in days rather than weeks. We will be screening aptamers for our target over the next few months. A number of companies are developing nanoparticle MRI contrast agents which could be very useful. In addition our experience in nanoparticle coupling will be very valuable in this project.

In summary:

We are looking nanoparticles as a means of delivering small molecules selectively to cancer cells. The initial work is being carried out by a "sandwich" student as there is some initial work to demonstrate the proof of principle.

Elizabeth Rapa, in her PhD and immediate post-doctoral work, demonstrated that it was possible to inhibit the spread of rhabdomyosaroma cells in a laboratory test system by using a small RNA molecule. The nanoparticle system might be a good way to deliver the inhibitory molecule selectively to the cancer cells in a patient, so that it had no effect on the normal functions of non-cancerous cells.

We have just finish writing up Elizabeth's work and will very shortly be submitting it for publication. Elizabeth was awarded her PhD in September 2008, and was in part supported by William's fund.

Over the next year we hope to develop the nanoparticle system further and fund a PhD student to work on it for a three year period. We hope that the student will be the "William Dodd Fellow", and that person will be very largely funded by William's Fund.

Research Update from Elizabeth Rapa - October 2008

I have recently completed my D.Phil in the department of Paediatrics at the University of Oxford under the supervision of Dr Chris Mitchell. The title of my thesis is The Characterisation of the Differences in Gene Expression between Rhabdomyosarcoma cells and myoblasts. The purpose of this project was to see if we could find any differences between cancer cells and normal cells. I focused on the childhood malignancy called Rhabdomyosarcoma (RMS) which accounts for 5-8% of childhood cancer. Two different types of RMS exist; one type is called embryonal RMS (ERMS) usually affecting young children and associated with a good survival rate, and the other type is called alveolar RMS (ARMS) affecting older children with a much poorer survival rate. One very obvious difference between these types of RMS is the development of metastases in patients suffering with ARMS. Metastasis describes the process where by some of the cancer cells have been able to “break away” from the primary tumour, circulate through the bloodstream and grow in other normal tissues elsewhere in the body.

The first part of my study was to look at general changes in the expression of genes in cancer cells and in normal cells called myoblasts (a type of early muscle cell). I used two techniques - called representational difference analysis and microarrays. In both these techniques the cancer cells and normal cells are labelled differently, for example with different colours, making it possible to compare the amount of a gene in each different cell type. In this way, we can identify genes that are found at higher levels in the cancer cells and at lower levels in the normal cells that may be important in how a cancer cell develops. The main findings from these experiments were the over expression of genes in the cancer cells responsible for increasing cell proliferation and division, and genes that enable the cancer cells to evade mechanisms that would stop such uncontrolled cell division. The results also highlighted which gene pathways and networks are affected in the cancer cells, again providing more clues about what drives a normal cell to become a cancer cell. In addition a group of closely related genes were only found in ERMS cells and not ARMS cells, so these genes could be used as markers to help us distinguish ERMS from ARMS.

Approximately 90% of all cancer deaths are due to metastases, and as mentioned before ARMS are often associated with metastatic spread. Therefore the second part of my project focussed on investigating genes involved in allowing a cell to travel around the body, settle in a tissue and then grow into another tumour. Two genes, called ELMO1 and NELL1, were found at much higher levels in ARMS cells compared to ERMS cells and normal myoblasts, and have previously been implicated in cell migration and invasion. Experiments were set up to show that if ELMO1 and NELL1 were put into a cell that does not normally express these genes, the characteristics of that cell changed. The cells were now able to migrate. Similarly, stopping the expression of ELMO1 in ARMS cells by ‘silencing’ this gene caused these cells to lose their ability to migrate. Consequently ELMO1 could be a potential therapeutic target in the treatment of ARMS.

Although I have now finished my PhD, we hope to write up and publish the results from our metastasis work. Also, with the appointment of a new Professor of Paediatric Oncology in Oxford, we hope that the research on rhabdomyosarcomas will continue and there are already plans to set up a William Dodd Fellowship for another PhD student.

Elizabeth Rapa, 9th October 2008

Update from Chris Mitchell - May 2007

Abnormal Genes in Children's Cancers

Everything about an individual - from the colour of their eyes to their fingerprint patterns - is encoded by genes, which themselves consist of varying lengths of DNA. Genes, in turn are packaged into chromosomes, which, given a powerful enough microscope and appropriate conditions can be seen within each cell as worm like structures. Each human has 23 pairs of chromosomes, half of each pair being inherited from their mother and the other half of each pair inherited from their father. By way of example, one of the pairs of chromosomes determines, amongst other things, the sex of the individual, with two XX chromosomes determining a female and the XY combination a male, reflecting the function of some of the genes on these particular chromosomes. When a cell divides, the number of chromosomes briefly doubles up so that the two new cells end up with the full set of 23 pairs of chromosomes.

Sometimes, during the division of a cell, the chromosomes break into two parts. The body has the necessary repair mechanisms to deal with this sort of damage but occasionally the process goes awry and the bits of broken chromosomes do not find their proper partners and end up on another chromosome. This type of abnormality is known as a chromosome translocation. The very first example of this sort of abnormality was first seen in a condition called chronic myeloid leukaemia, where the bits of chromosomes 9 and 22 get jumbled to produce what is known as the Philadelphia chromosome, after the city where it was first described in the early 1960s. Since then there have been a range of similar abnormalities described in a wide range of cancers. Our particular interest is focussed on the translocation between chromosomes 2 and 13 (written as "t(2;13)") seen in a childhood tumour called rhabdomyosarcoma, which is a tumour of striped muscle. At the actual breakpoint of the chromosome will be a gene - now in two parts. When incorrectly reassembled there are now two hybrid genes which will not be under normal control and which will be making abnormal proteins - proteins which must have some effects on the cell which result in what we recognise as cancer.

This abnormality was first described the late 1980s and is now known to be associated with a poor prognosis for the patients whose tumour contains it. From a scientific view, the useful thing about a translocation is that it enables one to pinpoint the genes that are involved, which in turn allows the examination of the pathways those genes would normally be a part of. In the case of the t(2;13) we know that the two normal genes involved are called Pax 3 (for Paired boX 3) and FKHR (for FoRKHead) and that they have normal functions in controlling the migration and maturation of skeletal muscle. One of the hybrid genes will contain the first part of Pax3 and tail end of FKHR, and the other hybrid will be the converse.

We have previously described a method by which the abnormal repair might take place. Over the past few years we have been working to understand the effects of the abnormal genes. We have been able to determine that the presence of these two hybrid genes leads to the abnormal regulation of a large number of other genes within the affected cell. We have chosen to focus on a few of these abnormally regulated genes and in particular on those genes that might determine the ability of a cell to migrate through the body.

During normal development of a fetus, for example, the muscle cells start in the centre of the body and migrate out into the developing limb buds to form, ultimately the muscles that are present in fully developed arms and legs. This process is akin to that which occurs when cancer cells spread through the body to form secondary tumours. Understanding this process might begin to provide the insights needed to develop treatment, which could control or prevent the spread of cancer cells.

Obviously there is a long way to go from a laboratory experiment where we can watch cells migrate through some gel, and be able to turn this ability off, to the situation we might hope for in the future where this knowledge can be used to develop a treatment that prevents the cancer cells from spreading. Nevertheless it is a start. The work is exciting to watch unfold, and we have been extremely grateful to William’s Fund for all the support they have given us over the past years.

Chris Mitchell
Paediatric Oncologist
Oxford
1st May 2007

August 2005 Report from Christopher Mitchell PhD FRCP, Consultant Paediatric Oncologist (formerly William's consultant)

It has been some time since we updated the report on the website, and there has, of course, been progress with our work over the past months. We now have two projects, the first and original one which is looking at some of the underlying genetic abnormalities in childhood connective tissue tumours, and the second, and much smaller project, which is examining similar mechanisms in childhood leukaemia.

We have made a lot of progress with the main project over the past months, following a lot of hard work and a number of setbacks. However, we have now reached a point where we can hope for substantial progress over the next few months as we analyse our new results and design new experiments to answer the questions that are now raised. It is a continuing process, and we remain a long way from understanding the “cause” of this kind of cancer.

The leukaemia project has already given some interesting insights into the underlying genetic mechanisms. This work is very much at a preliminary stage, but we hope to develop this project further over the next few years.

Project 1: Genetic mechanisms in childhood sarcomas; (personnel: Sophie Hill, PhD and Elizabeth Rapa MSc)

Sophie Hill who started working with us in March 2001, has continued to work part time in the laboratory since the birth of her second son. Elizabeth Rapa, who started work for us in March 2002 brought a great deal of experience in the molecular biology of cancer and has been a great addition to the team. She is now registered at Wolfson College , the University of Oxford for a Doctorate in Philosophy. Both Elizabeth and Sophie are working on a tumour called rhabdomyosarcoma, which is a type of cancer that develops in muscle cells and is seen mainly in children. Emily Carpenter continues to work intermittently in the lab, fitting this work around her medical studies and has concentrated on the genetic abnormalities that underlie the development of leukaemia.

These three appointments have been possible only as a result of the generosity of the friends and family of William Dodd. We are grateful to Peter and Johanna Dodd for this opportunity to thank all of you who have given so generously to support this research. The past year has at times been very frustrating in the lab, but now we have a lot of new information, which is at last enabling us to design new experiments that should yield some exciting answers about the underlying causes of this type of cancer.

First, we need to explain a bit about genetics. Everything about us is encoded in the chemical known as DNA. The DNA in our cells is organised into genes and the genes in turn are organised in chromosomes. The genes provide the blueprint for producing all the proteins that cells need to function. A human cell contains about 100,000 genes, organised into 23 pairs of chromosomes. In any one cell though, only a few genes relevant to that cell's function are turned on or "expressed". Obviously, it is important that the DNA is kept in proper condition while the cells do the work required of them or when they divide, so there are intricate mechanisms within the cell to make sure that any damage is properly repaired and that the chromosomes replicate themselves properly. These mechanisms are also encoded in the genes, so it is rather like having a car that can repair itself if it stops working properly, or can make itself a new one when the old one wears out!

Occasionally, however, when a cell is damaged or divides, the processes do not work properly and the chromosomes can get jumbled up - a process called chromosome translocation. For example, a bit of chromosome 2 can break off and get stuck onto a broken bit of chromosome 13. When this happens part of a gene on one chromosome adjacent to the break can find itself in the company of part of another gene adjacent to the break on the other chromosome. The two genes can then get spliced together to make a completely new gene with all sorts of unusual properties.

Many malignant tumours have within their cells recognisable and specific rearrangements of their chromosomes, and often it is possible to demonstrate that the affected genes have combined to make either a new protein or one that expressed when it shouldn't be. We are particularly interested in a translocation between chromosomes 2 and 13 seen in rhabdomyosarcoma. We think that the translocation results in one or more genes being turned on or off inappropriately. We are trying to understand how it is that such abnormalities of genetic control can happen, in the hope that we might in due course discover ways of re-establishing normal control. Such a discovery might in due course provide us with a completely new way of treating this type of tumour, and might also give us hints for dealing with other types of tumours.

With the support of William's Fund, we have worked to identify genes that are no longer regulated properly, presumably as a result of the chromosome translocation that we described above. The technique – called “representational difference analysis” - was difficult to master and prone to not working - usually for no very obvious or good reason. At last, the technique has been optimised and we have now completed a number of experiments, leading to the identification of a number of genes that are not regulated normally in rhabdomyosarcoma. We chose eight genes to study further, based on features such as involvement in other types of cancer or because they are known to have roles in normal cell division. We have now confirmed that the regulation of these eight genes is significantly different in our cancer cells compared to normal cells. The next steps will include experiments, for example, to show what happens when a gene that that is over-expressed in a cancer cell is “turned off”, and similarly what happens when a gene which is under-expressed is turned “back on”. This work is the basis of a thesis that Elizabeth is submitting for a Doctorate in Philosophy.

We are also continuing to work on disentangling the series of events in cells at different points during cell of division – called the cell cycle - so as to identify genes that are inappropriately turned on or off during this process. This series of experiments involves sorting the cells into different populations according to their stage of division. Then the cell messages (RNA) are allowed to “stick” to a “chip” which has sequences from thousands of genes embedded on its surface. Where the cell message finds its partner, it will stick and we can identify it. This method – which is fairly new – generates a great deal of data very quickly, and hence enables us to quickly plan more experiments for the future.

We are building up a complete picture of all the events that are going wrong in the cancer cell, but we still have a long way to go. We are preparing another grant application and are hopeful, after some encouraging comments from our last application, that this time we will be successful. As always, we are very aware that these grants are relatively small in number and extremely competitive. We remain extremely grateful to all of the supporters of William's Fund for giving us the opportunity to pursue our research.

Project 2. GATA1 mutations in childhood leukaemia. (Emily Carpenter BSc, Research Assistant (Temporary) supported by William's Fund)

I am a graduate entry medical student at Oxford University . Before deciding to embark on a career in medicine, I previously did a degree in biology. I am particularly interested in understanding the biology of cancer and how new-targeted therapies might improve the treatment of people with cancer. I have been very fortunate to receive support from William's Fund for some research work that I have been doing during my medical training.

I have been working with Dr. Chris Mitchell and Dr. Paresh Vyas at the John Radcliffe Hospital . Our work has focussed on improving our understanding of how acquired changes (mutations) in a particular gene (GATA-1), are involved in causing leukaemia in children. It is an important area of research because many advances have been made recently in our understanding of the biology of leukaemia, and these advances have the potential to make significant contributions to our knowledge of the general mechanisms of cancer formation.

To date I have been involved in work that has led to the publication of two research papers. This work was on looking for mutations in the GATA-1 gene in two children that had acquired three copies of chromosome 21 (trisomy 21) in their leukaemic cells. Normally each cell has only 2 copies of each chromosome (one from each parent), which carry genes encoded in DNA. This area was particularly interesting to study because the type of leukaemia the children had is called AMKL, which occurs at high frequency in children with Down syndrome. Down syndrome is known to be caused by the presence of trisomy 21 (i.e. three copies of chromosome 21) in all the cells of the body. However, it was unclear what role the trisomy 21 was playing in causing the leukaemia. Our work confirmed the presence of GATA-1 mutations in these two children with AMKL and trisomy 21 only in their leukaemic cells. This finding suggests that trisomy 21 plays a role in the development of leukaemia through its presence in the precancerous blood cells, rather than other cell types in the body.

This summer I have also been involved in a study looking for the presence of GATA-1 mutations in older children with Down syndrome. This work is very relevant area because a new chemotherapy trial is being carried out using lower doses of drugs in patients who have a GATA-1 mutation. The trial is being carried out because these children have much better chance of cure and respond better to lower drug doses than patients without the mutation. This work should lead to the publication of another paper in the near future.

I would like to thank William's Fund very much for the support I have received, without which I would not have been able to carry out this work. I really enjoy the opportunity to be able to do some research alongside my medical degree. I hope the experience this gives me means I will be able to continue to contribute to the field of research into children's cancers after I qualify as a doctor.

Download the above report

Read the Report from 2003

The Childhood Cancer Research Fund is part of the Oxford Radcliffe Hospitals Charitable Fund.

Message from Peter and Johanna Dodd

William' s Fund has been raising money for the Childhood Cancer Research Fund since June 2000. With your help we have raised thousands of pounds in memory of William and we would like to thank all of you for your contributions and help. We would like not only to thank everyone but to update you on what the money will be spent on and to explain a little of the vital work which will be carried out and our future plans.

The money raised has enabled the team at the John Radcliffe Hospital to employ a full-time postdoctoral research fellow to work on the childhood cancer project. Sophie Hill was recruited in 2001 and is in the employ of Dr Chris Mitchell, Consultant Paediatric Oncologist, who was responsible for William's treatment. To finance this research project costs £40,000 a year and we have so far been able to reach this level of financing year-on-year - thanks to all of you. Below is a letter from Dr Mitchell which is a personal 'thank you' from the team and also a more scientific explanation of the Childhood Cancer Research project.

Johanna and I have been quite overwhelmed by the kindness and support shown by all of you and we hope you will continue to support the fundraising events in the future. Some of you have held some fantastic fundraisers yourselves and for this we are grateful too! We now see this as a reasonably long-term project and we are hoping, in conjunction with the team at the John Radcliffe, to take this programme forward over the next 5 years.

We would like to thank you all again for your tremendous support - it means a great deal to us and the charity as at present they rely solely on charitable donations for their research. It has also been a boost to the morale of the Oncology staff at the Radcliffe.

In the meantime we intend to continue our fund raising activities as a fitting memorial to William, to provide support to the Childhood Cancer Research charity and the Oncology staff at the Radcliffe, who were so caring and supportive during William's treatment programme. We will continue to provide regular progress updates as appropriate.

With Sincere Thanks
Johanna & Peter Dodd

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