Leveraging circulating tumour DNA (ctDNA) to dissect the evolutionary genomic dynamics of drug resistance in prostate cancer.

1. Scientific/medical context of research?
Prostate cancer is the commonest male cancer (41,000 diagnosed in 2010) and the second commonest (10,700 died in 2010) cause of male cancer death in the UK. One man dies of prostate cancer every hour in the UK. The growth of the prostate is dependent on male hormones (androgens). Prostate cancer develops when prostate cells grow uncontrollably. This is initially dependent on androgens. If diagnosed early prostate cancer can be cured by surgery and/or radiotherapy. However, 30% of cases will relapse and more than 20% of cases will present with widespread (metastatic) disease that is incurable. Initial treatment strategies to lower hormone (androgen) levels provide robust responses in 90% of cases (hormone naïve prostate cancer). However, with time, cancers develop other mechanisms of resistance despite low (castrate) levels of testosterone - known as castration resistant prostate cancer (CRPC). Unfortunately, this advanced form of prostate cancer is currently incurable. Over the last two years, two novel treatments have become available as treatment options for CRPC along with chemotherapy. These agents are Abiraterone acetate and Enzalutamide. These agents have been proven to prolong survival in patients with CRPC. Eventually, prostate cancer progresses despite these new drugs and chemotherapy: a proportion of patients respond for a number of years and others progress very rapidly.

2. What is the research trying to achieve?
Understanding why these agents stop working or drug resistance requires an understanding of DNA changes (genomic) and identifying these changes over time. Treatments for prostate cancer can influence the growth of prostate cancer and understanding this would require analysis of multiple cancer biopsies. This is an impractical solution as performing multiple biopsies is difficult and more importantly uncomfortable to the patient. This proposal will aim to identify these abnormalities with a simple blood test that can be done regularly. This blood test will be able to serially/sequentially monitor tumour gene changes and identify abnormalities in the androgen receptor and other genomic changes that associate with resistance.

3. Why is the research important?
Better drugs and management strategies are urgently required. Understanding and identifying causes of resistance at different time points throughout the disease course will allow us to institute early treatment changes and therefore improve patient outcome. Additionally new drugs can be developed to target these mechanisms and also implement a personalised treatment approach for the patients using a non-invasive technique and identify the right treatment for the right patient groups and reduce unnecessary side-effects with ineffective treatments.

4. Who is carrying out the research?
Dr Anuradha Jayaram is a clinical research fellow in the Prostate Cancer Targeted Therapy group at the Royal Marsden Hospital in London. She completed her medical degree and specialist oncology training in Ireland. Her career ambition is to be an academic medical oncologist with a laboratory focused on translational research within the field of prostate cancer. This fellowship will be carried out under the supervision of Professors Mel Greaves, Dr Gerhardt Attard and Professor Ros Eeles who are key opinion leaders within the fields of cancer evolution, prostate cancer targeted therapy and prostate cancer genetics. Dr Sharp will be based in the Centre for Cancer and evolution at the Institute of Cancer Research, the top rated academic unit worldwide. The fellowship will initiate a consortium (sponsors and collaborators) of international leaders within the fields of circulating biomarkers and molecular characterisation in prostate cancer medicine to ensure the greatest scientific and clinical impact of this fellowship.

Aims/Objectives
1.To use high-coverage targeted next generation (NGS) on sequentially collected plasma DNA to:
a. sub-classify and characterise AR-aberrant versus AR-wild-type CRPC.
b. define the genomic evolutionary changes that associate with resistance.
2. To use whole genome sequencing (WGS) data from plasma to interrogate the sequence of genomic events following distinct treatment selection pressures
3. To functionally validate AR somatic point mutations and other novel aberrations that associate with resistance in AR wild-type CRPC and develop clinically relevant strategies to target them.
Methodology:
Aim 1: An expanded high-coverage (>1000x) customised targeted NGS with an optimised panel design will be used. Pre-amplification barcoding of DNA fragments (Agilent Halopex HS) to improve the sensitivity and specificity of established approaches will be explored. Sequential plasma samples will be studied to identify genomic aberrations that temporally associate with resistance..
Aim 2: WGS at 100-150X coverage optimised for plasma will be used to determine clonal diversity of ctDNA and the order of copy number changes.
Aim 3: Functional validation of AR mutations and novel targets will be performed in prostate cancer cell lines (LNCaP, VCaP, PC3). Novel targets will be qualified using clinically applicable assays (eg ddPCR) on independent test sets.
Scientific and Medical Opportunities
Plasma DNA analysis has the potential to improve patient outcomes by:
1. Molecular characterisation of men in real-time and sub-classification of CRPC into AR gain or no AR gain
2. Improving our understanding of the genomic drivers of treatment resistance in prostate cancer
3. Optimising new and existing treatments for prostate cancer by molecularly selecting patients using a minimally invasive blood test.

1. Commercial private sector beneficiaries
The targeted deep coverage next generation sequencing (NGS) and validation of novel aberrations from this proposal will be of potential interest to companies interested in developing circulating tumour DNA (ctDNA) based assays for diagnostics as well as companion biomarkers to help identify potential patients that will be suitable for a specific treatment and also predict treatment reposne. Currently, in prostate cancer, there is no companion biomarker to predict response to treatment. This will be of interest to drug companies.

2. Public Sector and Charities
Genomics England (GEl), part of the Department of Health has begun a flagship project of whole genome sequencing (WGS) of 100,000 NHS patients across rare diseases and cancer. Prostate cancer is part of the GeCIP (Genomics England Clinical Interpretation Partnership) domain. Currently in this programme, WGS is only performed on patients prior to therapy. Data generated from WGS from this project with sequential samples during treatment will be able to enhance the 100,000 project to select suitable key targets and also incorporate analytical strategies used to analyse changes with treatment over time. Potential collaboration between GEl and ourselves in the future will be mutually beneficial - in terms of understanding data generated from WGS as well as committing to findings to expand the genomics knowledge base.

Data generated from this proposal will also impact on the future research programmes of Prostate Cancer UK (PCUK) with regards to the utility of biomarkers and clinical validity and fund prospective research to clinically validate novel targets and patient stratification using a ctDNA based assay. It will also increase awareness with relation to new approaches in prostate cancer and spark academic interest for further research opportunities.

3. Policy Makers
Validation of novel targets as causes of resistance to conventional cytotoixcs or endocrine therapies using a ctDNA assay allows the development of a clinically-applicable, molecular, blood-based test for selection of the optimal treatment for individual, progressing CRPC patients. This will change the treatment paradigm and subsequently health service policies by introducing patient stratification for treatment selection based on selected biomarkers of response and resistance.

4. Prostate cancer patients and their families:
The fellowship and associated activities will impact upon prostate cancer patients and their families. The fellowship will aim to identify why a group of patients develop drug resistance to conventional cytotoxics and endocrine therapies using ctDNA, which is a simple non-invasive blood test. This will impact on patients' quality of life by stratifying patients to the right treatment and therefore sparing toxicities associated with treatments that are ineffective. More critically, it will identify therapeutic targets that improve the survival of this cohort of patients.