Lethal renal cell carcinoma sub-clones: Defining mechanisms of tumour evolution, treatment resistance and immune escape.

Kidney cancers have doubled in the UK over last 40 years, with >11,000 new cases annually and >4,200 deaths. Despite increased early detection the 5-year survival rates remain poor at 56%, with average survival of only ~18 months for advanced disease. Death is typically caused by disease spread to distal organs in a process called metastasis. While new drugs have been introduced to treat advanced metastatic disease, they are mainly used as palliative treatment options to delay rather than prevent mortality. This is due to drug resistance, which occurs in almost all patients, on average within 9 months. A major factor contributing to drug resistance is the extraordinary diversity found within tumours, caused by a pattern of continuous genetic mutation as the tumour grows. This means a tumour can actually be made up of many sub-tumours (called "subclones"), each of which is different. Treatment typically fails when some but not all of these "subclones" can be destroyed, and the ones left then grow back stronger causing terminal disease (called "lethal subclones").

This research will study how "lethal subclones" grow, how they spread across the body and how they resist drugs. The study will involve 320 kidney cancer patients, starting before drug treatment and will analyse DNA from their primary kidney tumour, which will be physically cut into a series of separate subregions (on average 7 per patient). This allows the different "subclones" to be looked at individually. As disease progresses and/or becomes treatment resistant, additional DNA will be analysed from tumours in other organs (metastases), from blood/urine samples as well as from autopsy tissue. This will allow the "lethal subclones" to be pinpointed, both before and after disease progression/drug resistance. By pinpointing the "lethal sub-clones" and tracking them through the disease course, it is anticipated that their strengths and weaknesses can be identified, generating fundamental biological knowledge to support the development of new treatment options. Another aim of this project is to develop a new cost effective diagnostic test to detect "lethal subclones", meaning high risk patients can be identified upfront and more aggressive treatment plans considered.

In the future a new generation of treatments called "immunotherapies" hold significant clinical promise to tackle "lethal subclones" and increase survival rates for kidney cancer. In other tumour types, such as skin, lung and blood cancers, breakthrough results have already been achieved in the last 5 years. The immune system plays an active role in kidney tumours, with white blood cells (lymphocytes) able to penetrate into the tumour and kill cancer cells. This process can be exploited therapeutically, either by raising the activity level of white blood cells or introducing more of them to attack the tumour. The great benefit of this approach, as compared to traditional drugs, is that the white blood cells are a living treatment that can adapt and keep up with the changing cancer cells. Immunotherapies have been previously tested in advanced kidney cancer patients with mixed results. A modest fraction of patients showed remarkable results (10+ years cancer free) however the majority failed to derive any benefit. The reasons for this are unclear, due to our limited biological understanding of how the immune system operates inside kidney tumours.

The second half of this project will conduct a detailed study of the immune system within kidney tumours. The DNA analysis from above will be complimented by RNA analysis and a technique called "multiplex immunohistochemistry", to map the location and activity of different types of white blood cell. This will be the biggest study of its kind in kidney cancer patients to date and aims to reveal how the immune system behaves in response to kidney cancer, providing insights to support the development of new immunotherapies.

Aim: To study the evolutionary dynamics of clear cell renal cell carcinoma (ccRCC), focusing on the subpopulations of tumour cells (subclones) that evolve to become metastatic, resistant to immune response, treatment resistant and ultimately "lethal".

Objectives:
1) To determine the features of lethal ccRCC sub-clones.
2) To develop novel scalable methods to detect lethal sub-clones.
3) To characterise the immune landscape of ccRCC and the mechanisms of lethal sub-clone immune escape.
4) To investigate the heterogeneity of immune gene expression in ccRCC sub-clones.

Methodology: Patients (n=320) will be recruited through the renal TRACERx study (PI=Prof. C. Swanton) with primary, metastatic & autopsy tumour tissue, together with circulating cfDNA, collected. High-throughput DNA sequencing will be conducted on a multi-region basis, combined with computational image analysis of tumour slides and statistical methods, to identify recurrent features unique to lethal sub-clones. Immunogenic profiling will be conducted using multiplex immunohistochemistry to phenotype tumour-infiltrating lymphocyte sub-types, RNA-seq for expression analysis and bioinformatics techniques combined with experimental validation (tetramer assay) to identify tumour-specific neoantigens. The functional mechanisms of immune gene expression will be investigated using methylation profiling and Capture Hi-C.

Scientific Impact: It is anticipated novel biological mechanisms of ccRCC tumour progression and treatment resistance will be identified. In addition new cost-effective methods, providing detail on tumour clonal architecture, will be piloted. Finally an increased understanding of ccRCC anti-tumour immunity, and how this is impacted by intratumour heterogeneity, is expected.

Medical Impact: i) biomarkers to guide ccRCC treatment, ii) novel diagnostic to identify patients with high tumour diversity, iii) immunogenic insights to support ccRCC immunotherapy development.

This project aims to advance our understanding of clear cell renal cell carcinoma (ccRCC) evolution, in particular the mechanisms of tumour progression, metastasis and treatment resistance. The project will go on to profile the immune landscape of ccRCC and its level of intra-tumour heterogeneity. The outputs from this work will impact the following groups:

1) Patients - Greater understanding of the causes of disease progression and treatment resistance offers direct benefit to ccRCC patients, through the discovery of biomarkers to better optimise treatment choice. In terms of new therapies to treat metastatic disease, clinical evidence suggests immunotherapeutic approaches have the most promising potential. Indeed, pre-existing evidence supports this with durable (>10 years) responses to cytokine therapy observed in a modest fraction of treated patients. The majority of ccRCC patients fail to derive benefit however, and the mechanisms underlying this are poorly understood. This project aims to complete the largest immunogenic study of ccRCC patient samples to date, and the biological insights from this could inform the design of future novel treatments. In particular, this dataset is unique, collecting information across multiple tumour regions and time-points, allowing therapies to be designed that can work in all possible tumour conditions. This fellowship will be completed in close collaboration with medical oncologists who actively treat ccRCC patients, meaning results will be translated as rapidly as possible. Clinically useful biomarkers, that can better inform therapeutic choice, may conceivably impact patients in a short-to-medium term timescale. Biological insights to support therapeutic development will impact patients on a medium-to-long term horizon.
2) Private Sector - The data generated in this project will impact the private sector by providing a rich resource to support ccRCC therapeutic development. The benefits realised include an ability to better prioritise therapeutic candidates, which can improve clinical outcomes and/or reduce wasted investment. Several small scale biotechnology companies, together with multi-national Pharmaceuticals, are engaged in ccRCC drug development. The results will be available to all private sector stakeholders through open access journal publications, and will benefit private sector in both the short and long term.
3) Scientists - Novel biological insights generated from this project will be published in open access scientific journals. This will benefit research groups working in a broad range of areas, providing knowledge to help inform future work. New methodologies generated from this project may enable other scientists to achieve more accurate or more efficient results than they can with existing methods. The data generated from this project will be made available as a resource for the scientific community, allowing further research questions to be addressed at no additional cost. The impact of these benefits will be realised within a short-time frame, with results being published and methods/data released as quickly as possible throughout the course of the fellowship.
4) Training - The applicant will benefit from Bioinformatics skills development, which will positively impact them throughout their career. The demand for well-trained informatics experts in cancer research remains strong, and so this training helps address a short supply in these skills.