Investigation of the mutational landscape of
Project leader: Dr Carmel McConville
Funding awarded: £15,000 joint funding CHECT and Fight for Sight; £14,200 CHECT
Project duration, start date: 1 year, May 2014
Project summary: Mutation of the RB1 gene is the primary event which initiates abnormal growth of developing retinal cells in the eye. However mutations (faults) in additional genes are also required for the formation of a retinoblastoma. Identification of these mutations, which may differ from one patient to another, and understanding how they influence retinoblastoma growth is important for the development of improved treatments.
In order to study these mutations in detail, it is necessary to have large numbers of retinoblastoma cells, more than can usually be obtained from a small piece of retinoblastoma tissue. Consequently we have developed retinoblastoma cell lines, which can be grown indefinitely in the laboratory, to provide large numbers of cells for this purpose. We have established cell lines from 15 different retinoblastomas and these represent one of the largest collections of retinoblastoma cell lines worldwide.
We will use the latest technology, known as 'next generation sequencing', to look at the DNA sequence of every active gene in these retinoblastoma cell lines. This sequence will be compared to a normal reference sequence in order to identify genes which are mutated. The sequence analysis will indicate which genes which are most frequently mutated in retinoblastoma and will also show how the overall cellular genetic activity is influenced by these mutated genes.
All of this information is essential to inform future research into the development of novel targeted therapeutics capable of reversing the effects of mutated genes in retinoblastoma.
Project status: In progress
Validation of Molecular Targets Characterizing Invasive Retinoblastoma
Project leader: Richard L. Hurwitz, MD, FARVO
Funding awarded: £39,756
Project duration, start date: Three years, May 2014
Project summary: The RB1 protein is a master regulator of cell growth. Changes (mutations) in the RB1 gene that codes for this protein are known to be the primary cause of most retinoblastomas (ocular tumours) detected in children and have also been found in common adult cancers such as lung, breast, prostate and ovarian tumours that have metastasized. Although almost all children with retinoblastoma have mutations in the RB1 gene, not all children have invasive disease, a precursor to the development of metastatic disease. We hypothesize that by comparing gene expression in tumours derived from children with invasive retinoblastoma to tumours derived from children with noninvasive retinoblastoma, we can gain an understanding of the metabolic steps after the mutation of RB1 that lead to tumour invasion and subsequent metastatic disease. We have studied gene expression in both invasive and noninvasive retinoblastoma tumours using next generation RNA-Seq technology and have identified 50 candidate genes that are more highly expressed in invasive retinoblastoma tumours when compared to noninvasive retinoblastoma tumours. We propose to validate these candidate genes by examining differences between non-invasive and invasive retinoblastoma tumours using in vitro and in vivo models of retinoblastoma as well as tumour samples from children. The advantages of studying retinoblastoma are 1) lack of complications caused by age or environmental exposures, 2) known molecular initiating events and 3) relatively rapid tumour initiation and progression. The results obtained will help stratify risk for metastasis, identify new therapeutic targets for retinoblastoma and improve approaches to genetic counseling of affected families.
Project status: In progress
Use of aptamers to increase the efficacy of HMGA2 targeted therapy in retinoblastoma
Project Leader: Dr S Krishnakumar
Funding awarded: £47,298
Project duration, Start Date: Two years, April 2014
Project summary: With advancements in molecular biology, targeted therapy is gaining importance in the treatment of RB. Aptamers have immense potential in targeted therapy because of their ability to bind to the target with high affinity and specificity. Previous findings show over-expression of the transcription factor HMGA2 in retinoblastoma transformation. Our previous work also showed the contribution of HMGA2 in cell proliferation and G1/S phase transition in Rb. HMGA2 was also found to regulate many oncogenic and tumor suppressive miRNAs in Rb. siRNA and Aptamer targeting HMGA2 were able to bring down the proliferation in Rb. But these biomolecules targeting HMGA2 should reach the tumor cells to modulate HMGA2 function with minimal uptake and damage to the normal cells.
In the present proposal, we would like to develop strategies for delivering the HMGA2 siRNA and aptamer,targeting HMGA2 to the retinoblastoma cells. Tumor cell specific delivery of the aptamer and siRNA sparing the non-cancerous cell would be the first parameter to be evaluated. We would also investigate that the delivery strategies do not compromise the affinity, stability and functional effect of the siRNA and aptamer targeting HMGA2. The two novel strategies proposed in this project are: delivery of HMGA2 siRNA through aptamer-siRNA chimeras into the retinoblastoma cells; and developing a novel antibody mediated delivery of aptamer into RB cells.
Project status: In progress.
Autonomic reflexes and cardio-respiratory instability during super-selective intra-arterial chemotherapy in the management of children with Rb.
Project Leader: Dr S P McGuirk et al. at Great Ormond Street Hospital and Birmingham Children’s Hospital.
Funding awarded: £18,000
Project duration , Start date: 1 year, planned start date 1 July 2013.
Project summary (as written by Dr S P McGuirk): Conventional management of retinoblastoma is associated with excellent long-term outcome. However, some children with advanced retinoblastoma do not respond to conventional management while others develop recurrent disease following initially successful treatment. Traditionally, the management of these children has involved external beam radiation or surgical removal of the eye. A number of new treatment modalities have been developed that can be used to treat children with advanced disease and also preserve the function of the affected eye. One of these new treatment options is super-selective intra-arterial chemotherapy. This involves the insertion of a small tube or catheter into the ophthalmic artery so that chemotherapy can be delivered selectively to the affected eye. The advantage of this technique is that it delivers a high concentration of chemotherapy to the tumour with few systemic side effects.
Previous studies have reported that intra-arterial chemotherapy is a safe and effective treatment in children with advanced disease. It causes complete regression of the tumour and preservation of vision in most cases. However, we have observed that a significant proportion of children developed severe adverse cardio-respiratory responses during the procedure. These reactions have occurred only during the second catheterisation procedure and are characterised by the sudden development of acute bronchospasm, impaired ventilation and marked haemodynamic instability. Patients have been resuscitated with intravenous fluids, intravenous atropine and intramuscular epinephrine with rapid restoration of normal cardio-respiratory function. None of the children had any adverse sequelae following these events.
We do not currently understand why these responses occur, why they occur in some children but not others, or why they are only clinically evident during the second catheterisation procedure. We believe that they represent an autonomic reflex response, which is qualitatively similar to the trigemino-cardiac and trigemino-respiratory reflexes observed during corrective surgery for strabismus, cranial and maxillofacial surgery, and skull base surgery. We propose to undertake this prospective case-controlled study to examine the pathophysiological events in detail. In particular, this study will determine the incidence of the trigemino-cardiac and trigemino-respiratory reflexes and quantitatively measure the effect of these reflex responses on haemodynamic and respiratory function. Ultimately, this information may allow us to identify adverse responses before they become clinically apparent, thus improving the safety of this procedure. The findings of this study are, therefore, of potentially immediate clinical application.
Project status: In progress. Link to article in Pediatric Anesthesia.
Role of SLRP family members in malignant progression of retinoblastoma.
Project Leader: Professor Shin-ichi Ohnuma Co-investigator: Dr Mandeep Sagoo
Funding awarded: £15,000 Joint funding applied for through CHECT and Fight for Sight
Project duration, Start date: 01/01/2013 for 1 year.
Retinoblastoma is the most common childhood cancer of the eye, affecting 1 in 20,000 live births, with 40-50 new cases diagnosed each year in the UK. Many children develop tumours that are aggressive and refractory to treatment, often requiring eye removal (enucleation), whereas some respond to local treatment and chemotherapy. Some years ago, it was reported that inhibition of a gene named as RB1 is important for initiation of retinoblastoma. However, recent studies have shown that the inhibition of RB1 gene is not sufficient to develop aggressive retinoblastoma and alternation of additional genes is required. However, little is known about additional genes that may be involved. If we can understand the detailed mechanism of retinoblastoma development, it might be possible to improve treatments.
This project will investigate the role of a family of genes which produce secreted proteins around the cancer cells. This family of genes are highly produced in the normal eye and known to be involved in formation of non-eye cancers. Moreover, our previous studies using cells isolated from retinoblastoma patients showed that amounts of some of the family members are altered in association with retinoblastoma development. Firstly, we will examine the change of gene expression in the process of retinoblastoma development using human retinoblastoma tissues. Then, we will examine their function on retinoblastoma progression using cells isolated from retinoblastoma tissues.
If we can prove the importance of the gene family in retinoblastoma, it becomes possible to develop new diagnosis and treatment of retinoblastoma.
Project status: Commenced April 2014 due to delay in ethical approval.
Understanding and overcoming barriers to living with an artificial eye
Project leaders: Tara Shea and Laura Rouse
Funding awarded: £14,339
Project duration, Start date:
Understanding the experiences of children and their families following enucleation will help to develop some clear best practice guidelines regarding clinical management of this population. We hope to promote innovation in the development of clinical resources to support adaptation to living with an artificial eye. By better understanding the scope of the barriers (physical, mental and emotional) that may be encountered by children and young people and their families who have experienced adjusting to living with an artificial eye, the research team can explore the support services and strategies used by children and their families who have undergone enucleation in order to address these barriers.
In order to understand the experiences of children and their families following enucleation the study will involve conducting interviews with three groups: children over five years of age who have experienced a unilateral enucleation; parents of children who have experienced enucleation; and teachers of children who have experienced enucleation.
It is anticipated that the findings of this study will increase our understanding of how to provide the most appropriate and research based strategies so that future families do not suffer further emotional distress relating to artificial eye problems.
Project status:Commenced April 2014 due to delay in ethical approval
Retinal vessel architecture in retinoblastoma patients - a predictor of progression?
Project Leader: Dr Clare Wilson
Funding award: £10,500
Project duration, Start date: Estimated start date 01/08/13
Project summary: (as written by Dr Clare Wilson)
Retinoblastoma treatment has developed leading to vastly improved survival rates. However, detecting early disease progression is challenging for experienced retinoblastoma surgeons who see over 20 new patients a year. It is even more problematic for surgeons working in countries with smaller populations. An objective assessment that can alert the surgeon to progression of the cancer would be of great benefit.
New software can measure changes in blood vessels at the back of the eye. Eye cancer doctors have made the clinical observation that these vessels change when disease worsens, and so could be used to detect eye needing more treatment. At The Royal London Hospital we have a large set of digital images from children who have had eye cancer and we are analyzing them with new software to see if the vessel changes can be used to accurately detect progression of the tumour.
If we get positive results, we could make the follow up visits for these children with eye cancer a more pleasant experience, yet just as accurate.
Project status: In progress.
Development of a next generation sequencing analytical pathway for improved test sensitivity and enhanced detection of low level RB1 mutations in retinoblastoma patients.
Project Leader: Dr Simon Ramsden Co-investigator: Dr Sarah Waller
Funding awarded: £13,810 Joint funding applied for through CHECT and Fight for Sight
Project duration, Start date: December 2013 for 1 year.
Mutations in the retinoblastoma gene RB1 cause the childhood eye cancer retinoblastoma. Traditional Sanger sequencing technology reliably detects mutations in RB1 in tumour tissue in the majority of patients from whom tumour is available, and detection of a germline mutation permits predictive testing of at risk family members. In our laboratory Sanger sequencing detects germline mutations in ~92% of patients with bilateral tumours and ~16% of patients with unilateral tumours.
Whilst Sanger sequencing offers appropriate test sensitivity for most cases there remain a small number of familial mutations undetected and also it has limited utility when trying to detect mosaicism. Emerging sequencing technologies (so called “Next Generation”) are showing great promise in both the academic and service environment and offer significant advantages over conventional Sanger testing. We feel that Next Generation Sequencing could offer us a means of overcoming both of these obstacles.
Project status: In progress.
Audit of clinical genetic and molecular genetic information and reproductive issues known to retinoblastoma families.
Project Leader: Dr Trevor Cole, Consultant Clinical Geneticist, Birmingham Women's Hospital
Funding awarded: £30,000
Project duration , Start date: 2 years starting in October 2008. The project overran and finished in late 2012, we await a final report.
Project summary (as written by Dr T Cole): The differing and complex genetics underlying the majority of unilateral and bilateral retinoblastoma means that many families have to be given genetic counselling advice on an ongoing and rather piecemeal basis.
We therefore suggest that we need to work with families currently undergoing treatment and, families discharged from follow up, to see if further genetic counselling input is necessarily to ensure families have the best information we are currently able to provide. We would suggest that this is important baseline data and would facilitate periodic review or audit that is likely to be necessary until all families are able to have a "definitive genetic explanation" to all at risk family members. Evidence on the practical benefits perceived by patients, in addition to the theoretical medical and cost benefits reported by Richter et al 2003, from molecular studies could then be assessed. This study would be timely as it could collect data prospectively from families yet to have definitive molecular answers and subsequent to these answers becoming available.
Progress report (As written by Dr T Cole): In early 2012 attempts to contact the remaining list of people was drawn to a close and we decided at this stage all remaining contact would be via letters to the participant’s GPs. Since this time several hundred further patient replies have been reviewed and letters to GPs are being produced and sent out to the remaining families.
Project status: Completed, awaiting final report.