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

Lead Research Organisation: The Francis Crick Institute
Department Name: Grants Administration

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The first findings from this award relate to the discovery that one particular type of tumour mutation, called a "frameshift indel", was found to be especially important in relation to how well patients respond to a new class of drugs called immunotherapy. Immunotherapies have led to complete disease regression and improved survival in a subset of cancer patients, notably in those with tumours of the kidney, lung or skin. A challenge that remains is working out which patients will benefit from these drugs. I studied the genetic mutations in detail across 200 melanoma patients, and found that 88% of patients with a high number of "frameshift indels" responded to immunotherapy, compared to only 43% of patients with a low number. The importance of "frameshift indels" is believed to stem from the fact they can cause long strings of altered DNA. Other mutation types (e.g. single nucleotide variation) typically change only a single letter of DNA code - this small scale change can be missed by the patient's immune system. However in the face of tens or hundreds of mutated letters it is the patients' immune system is much more likely to spot these errors and mount a potent immune response, leading to cancer cell destruction. An additional finding in my study was that unexpectedly kidney cancers had the largest number of "frameshift indels", more than either lung or skin cancers, which are typically among the most highly mutated tumours due to tobacco/UV exposure. This may be one of the contributing factors which explains why some kidney cancer patients respond exceptionally well to immunotherapy.
Exploitation Route In future research it is hoped "frameshift indels" can be used as part of a predictive diagnostic test, to better select which patients will benefit from immunotherapy. Work is already underway to test the feasibility of this approach. In addition the authors plan to explore whether frameshift indels can used therapeutically, as potent immune targets in tumour cell vacinations or adoptive immune cell-based therapies.
Sectors Pharmaceuticals and Medical Biotechnology

URL http://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(17)30516-8/abstract
 
Description Crick Idea to Innovation (i2i) Fund
Amount £70,000 (GBP)
Funding ID Project - 10646 
Organisation Francis Crick Institute 
Sector Academic/University
Country United Kingdom
Start 08/2017 
End 08/2018
 
Description Exploring cancer mutations as a preventative vaccination target, in individuals with high cancer risk from environmental exposure
Amount £237,000 (GBP)
Funding ID A2347 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 02/2023
 
Description Immune dysfunction as a biomarker for the early detection of lung cancer development and relapse
Amount £100,000 (GBP)
Funding ID C69256/A30194 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 03/2021
 
Description Investigating melanoma metastases
Amount £680,357 (GBP)
Funding ID Unknown 
Organisation Melanoma Research Alliance 
Sector Charity/Non Profit
Country United States
Start 01/2021 
End 12/2023
 
Description Next generation IO targets
Amount £260,628 (GBP)
Organisation Cancer Research Technology (CRT) 
Sector Private
Country United Kingdom
Start 11/2020 
End 12/2021
 
Title Representative-Sequencing dataset 
Description While thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g. 6mm-biopsy), which becomes grossly under-powered as tumor volume scales. Here we demonstrate Representative-Sequencing (Rep-Seq), as a new method to achieve unbiased tumor tissue sampling. Rep-Seq utilizes fixed residual tumor material, which is homogenized and subject to next generation sequencing. Analysis of intra-tumor tumor mutation burden (TMB) variability shows a high level of misclassification with current single biopsy methods, with 20% of lung, and 52% of bladder tumors, having =1 biopsy with high-TMB, but low clonal-TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact This data offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility. 
URL https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3404257
 
Title TRACERx Renal 100 Sequencing Dataset 
Description Tumour sequencing data from 1209 primary tumour regions, from 101 patients, was generated as part of this grant, and this data has been deposited to the European Genome phenome Archive (EGAS00001002793) 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact The results from this research were published in Cell. 
URL https://ega-archive.org/studies/EGAS00001002793
 
Description Early detection collaboration with Dr Richard Lee 
Organisation Royal Marsden Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution Myself and Dr Lee have recently jointly been awarded a CRUK Early Detection Primer Award, to investigate immune dysfunction as a biomarker for tumour development and progression.
Collaborator Contribution Dr Richard Lee - Consultant physician in respiratory medicine and champion for early cancer diagnosis at the Royal Marsden Hospital. Dr Lee is an expert clinician in respiratory medicine and early diagnosis of lung cancer. Dr Lee leads on the early diagnosis strategy at the Royal Marsden Hospital, and is developing an innovative range of clinical services and trials to support increased rates of lung cancer early detection including his role as joint clinical lead for the NHS England national lung cancer screening pilot. Within this project Dr Lee will lead the set-up and recruitment efforts to develop a new cohort of pre/non-malignant samples from patients undergoing longitudinal management for pulmonary nodules.
Impact Myself and Dr Lee have recently jointly been awarded a CRUK Early Detection Primer Award, to investigate immune dysfunction as a biomarker for tumour development and progression.
Start Year 2019
 
Description Immunology collaboration with Dr Sergio Quezada 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution This is an academic collaboration with Dr Sergio Quezada, world expert in anti-tumour immunology. I have conducted bioinformatic analysis, related to Fc-gamma receptor (FCGR) function, and the influence of human germline FCGR polymorphisms in melanoma patient response to immunotherapy.
Collaborator Contribution Laboratory work related to Fc-gamma receptor (FCGR) function, and other tumor immunology projects.
Impact Paper accepted with Cancer Cell, titled: "Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies".
Start Year 2017
 
Description Immunopeptidomics collaboration with Prof. Yardena Samuels 
Organisation Weizmann Institute of Science
Department Laboratory of Body and Mind Immunology Weizmann
Country Israel 
Sector Academic/University 
PI Contribution A collaboration has been initiated with Prof. Yardena Samuels at the Weizmann Institute in Israel. Through this collaboration I will be trained in a new technique called immunopeptidomics, a state of the art method for neoantigen identification. I will be spending a month in Israel in May-2019 training in the method. I am contributing tumour tissue samples from the TRACERx study.
Collaborator Contribution A collaboration has been initiated with Prof. Yardena Samuels at the Weizmann Institute in Israel. Through this collaboration I will be trained in a new technique called immunopeptidomics, a state of the art method for neoantigen identification. I will be spending a month in Israel in May-2019 training in the method. Weizmann is contributing methodological knowledge and expertise.
Impact Training skills will be the output, delivered in 2019
Start Year 2018
 
Description Representative Sequencing Project 
Organisation Roche Pharmaceuticals
Country Global 
Sector Private 
PI Contribution I have worked collaboratively with Roche/Ventana to develop a novel tumour sequencing approach called "Representative Sequencing". My contribution has been to perform bioinformatic analysis, and importantly give scientific input/direction to the project.
Collaborator Contribution Roche/Ventana have performed the laboratory experiments, and sequencing work.
Impact I presented an oral conference presentation at the annual 2018 Tucson Symposium related to this project.
Start Year 2017
 
Title Frameshift indels as a therapeutic target 
Description The technology is based on recent research findings arising from my fellowship, centred on the role of frameshift-insertion/deletion mutations (fs-indels) in tumour immunogenicity (1). The technology represents a therapeutic method that targets fs-indel derived neoantigens, as a cancer immunotherapy treatment. This technology is based contained in (1), which imply targeting fs-indel neoantigens specifically as opposed to single-nucleotide variant (SNV) derived neoantigens will give rise to superior efficacy. 1) Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis. Turajlic S, Litchfield K, Xu H, Rosenthal R, McGranahan N, Reading JL, Wong YNS, Rowan A, Kanu N, Al Bakir M, Chambers T, Salgado R, Savas P, Loi S, Birkbak NJ, Sansregret L, Gore M, Larkin J, Quezada SA, Swanton C. Lancet Oncol. 2017 Aug;18(8):1009-1021. doi: 10.1016/S1470-2045(17)30516-8. Epub 2017 Jul 7. 
IP Reference P112425GB 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact A company called Achilles Therapeutics has requested a license to the patent - negotiations with the Francis Crick Institute commercial team are underway.
 
Title Frameshift indels as a treatment biomarker 
Description The technology is based on recent research findings arising from my fellowship, centred on the role of frameshift-insertion/deletion mutations (fs-indels) in tumour immunogenicity and checkpoint inhibitor (CPI) response (1). The technology represents a predictive test/biomarker that uses the number (or fraction) of fs-indels within a tumour to predict response rates to CPIs such as ipilimumab, nivolumab and pembrolizumab, wherein a high number fs-indels compared to a reference sample indicates increased chance of drug response. 1) Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis. Turajlic S, Litchfield K, Xu H, Rosenthal R, McGranahan N, Reading JL, Wong YNS, Rowan A, Kanu N, Al Bakir M, Chambers T, Salgado R, Savas P, Loi S, Birkbak NJ, Sansregret L, Gore M, Larkin J, Quezada SA, Swanton C. Lancet Oncol. 2017 Aug;18(8):1009-1021. doi: 10.1016/S1470-2045(17)30516-8. Epub 2017 Jul 7. 
IP Reference P112332GB 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact The Crick translation team are exploring licensing opportunities, in discussion with pharmaceutical/diagnostic companies.
 
Title Molecular profiling as a clinical decision aid for patients with clear cell Renal Cell Carcinoma (ccRCC) 
Description A new method to stratify patients for surgery or drug treatment, based on an evolutionary classification system using tumour sequencing data. 
IP Reference P113326GB 
Protection Patent granted
Year Protection Granted 2018
Licensed No
Impact This invention has potential to help optimise patient treatment, leading to improved clinical outcome.
 
Description CRUK Research Fundraisers Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact I gave a lay talk to ~100 CRUK fundraising staff, who lead CRUK's work engaging with individual high profile donors. I presented an overview of the principles of tumour evolution, and an update on work from the TRACERx study.
Year(s) Of Engagement Activity 2017
 
Description Talk at University College London Hospital Clinical Trials Unit team building day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact ~150 clinical trial unit team members attended my talk, and were highly engaged and interested in the content. Several members followed up to find out more information.
Year(s) Of Engagement Activity 2019
 
Description Talk to national team of research nurses 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I gave a 30 minute lay talk to a national team of CRUK Nurses, explaining principles of kidney cancer evolution and the transnational benefits from this research.
Year(s) Of Engagement Activity 2019