Clonal heterogeneity and therapy resistance in Burkitt lymphoma

Burkitt lymphoma is a rare aggressive cancer of the lymph glands affecting approximately 200 people per year in the UK. Current treatment can be very successful, but this involves intensive chemotherapy and antibody therapies. For some people this type of treatment, carrying the greatest chance of cure, is too toxic and cannot be given. The outcome for these patients is much less good. For those people, both children and adults, able to tolerate the more intensive treatments, there is a high risk of severe infections and patients often have to spend long periods in hospital receiving strong antibiotics to fight infection, painkillers and support with feeding. Unfortunately, for patients in whom the lymphoma relapses there are few additional therapies and very limited chance of cure.

Currently, very little is known about why Burkitt lymphoma relapses. We do not fully understand the genetic changes which make a patient's lymphoma more likely to come back and we do not understand why it is that relapsed Burkitt lymphoma tends to be unresponsive to second-line therapies. Importantly, we have no information about how initial treatment with chemotherapy causes, or selects out, chemotherapy resistant cells which are then able to grow and cause a relapse.

This fellowship will develop our understanding of the natural variability which exists in a patient's lymphoma at the point when they are diagnosed and then track how this changes, or evolves, in response to chemotherapy treatment. These two factors are believed to be important both for a patient's chance of suffering a relapse and in determining the nature of the relapse, for example whether it might respond to different chemotherapies.

As Burkitt lymphoma is a rare cancer and, overall, patients have a good chance of cure, there are limited numbers of relapsed Burkitt lymphoma samples with which to carry out such work. In order to address this problem, we have been developing a collection of patients' samples, taken at diagnosis of Burkitt lymphoma, which we are able to study using mice with an immune system which has been altered to allow them to accept and grow the human lymphoma. These immune deficient mice are currently the only way to keep the lymphoma cells alive long enough to study how they respond to simulated chemotherapy treatment. Mice which have been transplanted with a patient's lymphoma cells will be treated with similar but less intensive chemotherapy to that used in humans. As the most intensive therapies are not used, these mice will eventually develop chemotherapy resistant lymphoma, and we are then able to study the differences between this model of relapsed disease and the initial sample taken from the same patient.

Patients' samples will be studied by looking at how the genetic variability, known as tumour heterogeneity, affects the chance of developing therapy resistance and how that heterogeneity changes over time when chemotherapy is given. The effect of chemotherapy on the behaviour of the cells and especially on which parts of a cell's biological machinery, known as its transcriptome, are turned on or off. Initially this will be done by looking at whole tumours from individual patients and mice, but more recent technological developments mean that we will also be able to look at a patient's tumour one cell at a time.

By understanding the factors which drive the risk of relapse it will be possible to more accurately predict those patients in need of aggressive therapy and those that may be adequately treated with less intensive treatments. Such an approach may open up potentially curative treatments to older or less fit patients who would currently not be eligible for curative chemotherapy. For patients who relapse, understanding what drives their therapy resistance will allow the rational design of new treatment strategies with a greater chance of cure.

Burkitt lymphoma (BL) is a rare non-Hodgkin lymphoma with a good chance of cure. However, this requires the use of intensive multi agent chemo-immunotherapy and is associated with significant acute toxicity, prolonged in-patient stays and the need for broad supportive care measures. Furthermore, many older adults are not suitable for such intensive therapy and their outcome is worse. For patients of any age who relapse, cure is very unlikely as disease is commonly resistant to multiple agents.

Little is known about the cellular, genetic or transcriptomic factors driving relapse. This fellowship will investigate the heterogeneity of tumours at diagnosis and how clonal, mutational and transcriptomic changes occur under therapeutic pressure and contribute to therapy resistance. To achieve this, I have developed patient-derived xenograft (PDX) models of BL suitable for treatment with multi-agent chemotherapy, analogous to that used initially in humans. Without treatment escalation it is predicted that these models will develop therapy resistance, as occurs in patients. Untreated and chemo-resistant models will be characterised by exome, targeted mutation and RNA sequencing and their clonality assessed by sequencing of the clonally unique V(D)J region. Evolutionary modelling will be conducted in collaboration with the ICOS computing group, Newcastle. Integrated pathway and network analyses will determine the clonal and transcriptomic drivers of relapse.

Drivers of relapse, identified in PDX models, will be validated using samples from therapy resistant patients and functionally examined in vitro. Therapeutically tractable targets will be assessed in pre-clinical studies using PDX models of relapsed disease, in collaboration with the CRUK Drug Development group in Newcastle. An improved understanding of factors predisposing to relapse or causing therapy resistance will allow patient stratification and guide the development of future relapse treatment strategies.

Successful delivery of this Fellowship will lead to a greater understanding of the risk of relapse and the drivers of therapy failure in Burkitt lymphoma. By understanding the processes driving therapy resistance, new approaches to treating relapsed Burkitt lymphoma can be developed. Not only will this improve the cure rates for patients whose lymphoma relapses, but a realistic chance of cure following relapse forms a pre-requisite to the development of clinical trials offering risk stratified dose reduction at first presentation. The potential for dose reduction will play an important role in the design of more tolerable therapies for older and frailer patients and may also be relevant for patients suffering from endemic Burkitt lymphoma in low and middle-income countries, which are unable currently to deliver intensive chemotherapy regimens.

This application will bring teams managing paediatric and adult non-Hodgkin lymphoma together to help to address challenges which are equally important in both settings. This will represent an early opportunity to collaborate across the age groups, inclusive of the frequently under represented teenage and young adult population, based on our existing understanding of the molecular similarities of sporadic Burkitt lymphoma throughout life. Due to the rarity of the disease, age independent findings from this fellowship would support the concept of early phase trials spanning paediatric and adult age groups. This could make the UK a more attractive site for commercial investment into early phase clinical trials.

The development of pre-clinical models carries two possibilities for commercial development. Putative therapeutic targets which have been validated, will be developed in conjunction with Newcastle Drug Discovery and Cancer Research UK Commercial Partnership teams. The PDX models developed will be made available for academic or industrial collaborations, but will also be considered for commercialisation through collaboration with contract research organisations, broadening their availability into a global scale.

Development of the research team delivering this work in an important component of the fellowship's success. A recent Royal College of Paediatrics and Child Health report on the change in child health research in the UK identified a relative lack of senior lecturer level clinical academic paediatricians (Turning the Tide: five years on, RCPCH, 2018). This Fellowship forms a critical component of my transition to independence as a child health researcher. Furthermore, the UK Government's commitment to incorporate genomic medicine into mainstream clinical care will require clinical academic knowledge and understanding across specialties to facilitate integration into existing practice. For me, increased understanding and experience with bioinformatic analyses, including integrative network analyses and analysis of single cell data are important personal and strategic goals. Embedding these approaches within lymphoma research in Newcastle will strengthen our basic and translational potential. Delivering cutting-edge research will also help to foster relationships with new and existing collaborators across Europe, an essential component of maintaining UK relevance following the EU withdrawal. This will also be supported by the developmental aspect of the post-doctoral position within this Fellowship, developing interdisciplinary working with computer science colleagues and increasing world class bioinformatics capacity within Newcastle and the UK.

Finally, this Fellowship will raise the profile of tumour heterogeneity and clonal evolution within taught courses in Newcastle, allowing me to integrate new knowledge into lectures and linking internationally leading research and teaching excellence.