Evaluation of the CD4+ T cell tumour microenvironment of low grade B cell lymphoproliferative disorders

This work will look at the tumour microenvironment of subtypes of low grade B-cell non-Hodgkin's lymphoma, the 6th most common cancer in the UK and 4% of the total cancer burden. The tumour microenvironment, including both the cancer and immediate surrounding tissue, is made up of both lymphoma cells (the name given to cancer cells in non-Hodgkin's lymphoma) and other cells, particularly those of the immune system such as T cells. While researchers and clinicians now realise that how a cancer behaves is not entirely explained by the cancer cells themselves, it still remains unclear exactly how the overall tumour microenvironment might influence cancer behaviour, including growth rate and response to treatment.

The work will use advanced microscope-based techniques to look at the tumour microenvironment in lymphoma biopsies that have been obtained from patients. These biopsies are taken at the time of a patient's initial presentation with the disease and if and when the patient relapses. Previous work indicates that T cells (specifically those known as CD4+ T cells, of which there are a number of different types) may promote the progression of these diseases, both directly and by inhibiting the immune system's normal ability to kill cancer/ lymphoma cells. This project will seek to help answer the question whether T cells are responsible for many features observed in day to day clinical practice. This would include rate of progression, determination of when patients need treatment, the nature of the response to current standard treatments given in the clinic today and how it may influence newer therapies that are becoming available (often in clinical trials) or being developed.

This project has four primary objectives: 1. Develop a pipeline of advanced microscopy techniques to describe the tumour microenvironment in patient biopsy samples, especially with respect to the CD4+ T cells present; 2. Relate microscopy data to what we already know about the patients' samples, both with respect to other laboratory based tests and the overall clinical picture; 3. Determine whether we can predict how different patients respond in different situations both in clinical trials and in everyday care; and 4. Help the wider lymphoma research community ask relevant new questions about these diseases.

The pipeline will include the staining of biopsy samples with specialised markers to identify different cell types. We will then use different microscopy and imaging techniques to generate data that can then be analysed by computer algorithms. This will describe in very specific detail the different type of CD4+ T cells present, their number, their status and their location. Further, this analysis will generate information about how these CD4+ T cells interact with any lymphoma cells present and other cell types in the microenvironment.

We are particularly interested in relating our findings to what we already know about patients, both in terms of any other investigations done (both in a clinical and research setting) and how the disease is behaving in the clinic. We also want to use this work to develop tests that will help decide what type of treatment a patient might need when they develop lymphoma. Ultimately, we anticipate that this work will result in a more individualised approach to treating patients, including what treatments to use and when.

This proposal will examine the role that CD4+ T cells have in promoting tumour progression in B-lymphoproliferative disorders. In these diseases an inflammatory pro-tumoural tumour microenvironment (TME) exists, with resultant division and growth of lymphoma cells. Assessment of numbers, co-occurrence, location and spatial relationships of cells and cell clusters within the TME of these diseases has to date been restricted by generally small biopsy material available from routine clinical samples and the limitations of conventional immunohistochemistry. Advanced microscopy-based techniques include imaging mass cytometry and multi-colour immunofluorescent confocal microscopy. These two techniques are complementary: the former provides the ability to determine the anatomical inter-relationship of deeply phenotype tissue at lower resolution, the latter providing high resolution but limited parameter information. This project will use these two techniques in conjunction to explore the hypothesis that CD4+ T cells are drivers of B-lymphoproliferative disorders. Key research themes and questions to answer include: 1. How to optimise an advanced microscopy-based immune mapping pipeline to deep phenotype CD4+ T cells and their interrelationship with lymphoma and non-neoplastic cells, 2. Correlation of these findings with biological and clinical features and, 3. How to apply these analyses to inform related pre-clinical and clinical research and as a clinical bioassay. The pipeline development will necessitate appropriate primary patient sample selection followed by optimisation of panel design for CD4+ subset identification within the TME, image generation using both MCIF and IMC, and development of a bespoke analysis platform using established computer algorithms to perform cell segregation, neighbour analysis and pixel by pixel analyses. The output from the pipeline will give a tool that can be used for pre-clinical and clinical downstream applications.

The non-academic beneficiaries of this research are identified as follows:

1. Policy makers. These will include national leaders within the national cancer research institute (NCRI) clinical studies groups (CSG), who determine the strategy for the UK by the clinical trial portfolio and engagement with regulators, funders, professional bodies including NICE and Royal Colleges/disease societies.
2. Industry beneficiaries. These will primarily be those involved in developing novel therapeutics in B-cell non-Hodgkin's lymphoma.
3. Practitioners. This will include the wider clinical community, including medically qualified haematologists, trainees, clinical nurse practitioners and allied health professionals
4. Patients, particularly those with B cell non-Hodgkin Lymphoma including Chronic Lymphocytic Leukaemia/Small Lymphocytic Lymphoma, Follicular Lymphoma, Marginal Zone Lymphoma and related low grade B cell lymphomas.
5. The wider public.

The primary purpose of this proposal is to use routinely obtained patient biopsy samples in patients with lymphoma to generate a systematic way of evaluating the tumour microenvironment in patients with lymphoma. Along with other analyses (e.g. genomics), a clear understanding of the microenvironment is needed to improve our understanding of the underlying disease process, the prognosis that the disease has and how to make decisions about therapies. Unfortunately, however, current cancer treatments generally continue to offer a "one size fits all" approach, despite an increasing array of different types of treatment being licenced by the regulatory bodies. The traditional clinical trial where one treatment is compared to another, with subsequent proof of effectiveness of the experimental arm over the standard arm, can take years to complete. Patient selection for such trials continues to be in broad subtypes, with little risk stratification. Licencing and subsequent funding of new treatments as an outcome of these trials remains slow. The public purse is therefore stretched by offering untargeted and therefore ineffective expensive treatments. The improved understanding of disease progression and response to treatment will benefit patients, clinical practitioners, and industry researchers working in new therapeutics. The wider research and policy community will benefit from our overall improved understanding of the microenvironment in lymphoma as well as other cancer disease systems.

Timelines for non-academic impact will be mainly following completion of the initial phase of objective 1, throughout the remainder of the 3-year proposal and in the following years. Therefore, full engagement with practioners, patients and public is envisaged as soon as the initial milestone of optimising the tumour microenvironment pipeline is achieved to explain the output of our research and how it can be utilised in the future in improving our understanding of the disease and better designs for future therapies. At this same stage, engagement with policy makers and industry will begin in earnest to actually implement change. Continued engagement will then continue throughout the remainder of the proposal period and after.

Overall, by effective impact with all of the stakeholders in this process, the research proposed here is more likely to be better realised through better risk stratification of patients, appropriate delivery of current therapeutics, better design of future therapeutics and overall adding to the individualised medicine of the future.