Hypoxia-driven Prostate Cancer Genomics (HYPROGEN) - Illuminating the Genomic Landscape of Hypoxia-driven Early Metastatic Prostate Cancer
Conditions: Prostate Cancer, Hypoxia
Sponsor: The Christie NHS Foundation Trust

 
 
Hypoxia-driven Prostate cancer 
Genomics (HYPROGEN) 
Illuminating the genomic landscape of hypoxia-driven 
early metastatic prostate cancer 
 
Chief investigator:  
 
Prof Robert G Bristow, MD PhD 
x 
IRAS No.: 
 
 
262789 
x 
REC No.: 
 
 
19/NW/0569 
x 
Sponsor: 
 
 
The Christie NHS Foundation Trust 
x 
Sponsor Ref. No. 
 
CFTSp155 
x 
Christie Ref No. 
 
18_DOG04_201 
x 
Funder: 
 
 
The University of Manchester 
 
Protocol Version History 
Version 4.0 
08.04.2022 
Version 3.0 
10.09.2021 
Version 2.0 
05.11.2020 
Original Protocol v1.0 
12.07.2019 
550 Wilmslow Road
Withington
Manchester
M20 4BX
Switchboard: 0161 446 3000
Web: www.christie.nhs.uk
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SIGNATURE PAGE 
HYPROGEN: HYpoxia-driven PROstate cancer GENomics 
This document describes the HYPROGEN study and provides information about 
procedures for entering patients into it. The protocol should not be used as a guide for 
the treatment of patients outside of the study.  
The undersigned confirm that the following protocol has been agreed and accepted and 
that the Chief Investigator agrees to conduct the study in compliance with the approved 
protocol and will adhere to the principles outlined int the ICH Good Clinical Practice 
guidelines, the Data Protection Act (2018), the Declaration of Helsinki, Ionising Radiation 
Medical Exposure Regulations (IRMER), the Human Tissue Act (2004), the Research 
Governance Framework (2005)WKH6SRQVRU¶V623 and other regulatory requirement. 
The undersigned confirm their agreement to ensure that the confidential information 
contained in this document will not be used for any other purpose other than the 
evaluation or conduct of the investigation without the prior written consent of the 
Sponsor. 
The undersigned also confirm that the findings of the study will be made publicly 
available through publication or other dissemination tools without any unnecessary delay 
and that an honest accurate and transparent account of the study will be given; and that 
any discrepancies from the study as planned in this protocol will be explained. 
Every care was taken in drafting this protocol, but corrections or amendments may be 
necessary. These will be circulated to all Investigators in the study as required. 
For and on behalf of the Study Sponsor: 
Signature:  
.............................................................................................. 
 
Date: 
....../....../...... 
Name (print): 
.............................................................................................. 
 
 
Position: 
.............................................................................................. 
 
 
 
Chief Investigator: 
Signature: 
.............................................................................................. 
 
Date: 
....../....../...... 
Name: (print): 
.............................................................................................. 
 
 
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HYPROGEN - Hypoxia-driven metastatic prostate cancer genomics 
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KEY STUDY CONTACTS 
Chief Investigator 
Prof Dr Robert G Bristow, PhD; Director, Manchester Cancer 
Research Centre; Co-Director, CRUK Manchester Institute; Chief 
Academic Officer & Honorary Consultant, The Christie NHS 
Foundation Trust; University Professor of Cancer Studies, The 
University of Manchester; 
Email: robert.bristow@manchester.ac.uk  
Co-Investigators &  
Sub-investigators 
Dr Martin Swinton, Clinical Research Fellow, The Christie NHS 
Foundation Trust;  
Email: m.swinton@nhs.net 
Tel: 07896026629 
 
Dr Christoph Oing, Research Fellow, Translational 
Oncogenomics Group, Manchester Cancer Research Centre; 
Honorary Clinical Fellow Medical Oncology, The Christie NHS 
Foundation Trust;  
Email: christoph.oing@manchester.ac.uk 
 
Prof Ananya Choudhury, Chair and Honorary Consultant Clinical 
Oncologist; The Christie NHS Foundation Trust; 
Email: Ananya.Choudhury@nhs.net 
 
Professor David Wedge 
Manchester Cancer Research Centre 
The University of Manchester 
Email: David.Wedge@manchester.ac.uk 
 
Dr Pedro Oliveira, Consultant Histopathologist, Dept of 
Histopathology, The Christie NHS Foundation Trust; 
Email: Pedro.oliveira1@nhs.net  
 
Dr Andrew Hudson, Consultant Clinical Oncologist; The Christie 
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NHS Foundation Trust; 
Email: andrew.hudson8@nhs.net 
 
Dr Ben Taylor, Consultant Radiologist, Dept. of Radiology, The 
Christie NHS Foundation Trust; 
Email: ben.taylor19@nhs.net 
 
Dr Jon Bell, Consultant Radiologist, Dept. of Radiology, The 
Christie NHS Foundation Trust;  
Email: jon.bell2@nhs.net 
Dr Damian Mullan, Consultant Radiologist, Dept. of Radiology, 
The Christie NHS Foundation Trust;  
Email: damian.mullan@nhs.net 
 
 
Prof Noel Clarke, Director GU Research Group Paterson Institute; 
Director MCRC Pan-Manchester Biobank, The Christie NHS 
Foundation Trust; 
Email: noel.clarke@nhs.net 
 
Prof Vijay Ramani, Consultant Urological Surgeon; The Christie 
NHS Foundation Trust; 
Email: v.ramani@nhs.net 
 
Mr Jeremy Oates, PhD, Consultant Urological Surgeon; The 
Christie NHS Foundation Trust; 
Email: jeremy.oates@nhs.net 
 
Mr Maurice Lau, Consultant Urological Surgeon; The Christie 
NHS Foundation Trust; 
Email: maurice.lau@nhs.net 
 
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Mr Satish Maddineni, Consultant Urological Surgeon; The 
Christie NHS Foundation Trust; 
Email: satish.maddineni@srft.nhs.uk 
 
Dr Maria Joao Serra, Consultant Clinical Oncologist; The Christie 
NHS Foundation Trust; 
Email: maria.serra@nhs.net 
 
Dr YeePei Song, Consultant Clinical Oncologist; The Christie NHS 
Foundation Trust; 
Email: yeepei.song1@nhs.net 
Study Co-ordinator 
Dr Martin Swinton Clinical Fellow, The Christie NHS Foundation 
Trust;  
Email: m.swinton@nhs.net 
Tel: 07896026629 
Sponsor Representative 
Mr Wes Dale, Managing Director ± Research, Innovation and 
Education, The Christie NHS Foundation Trust; 
Email: wes.dale@nhs.net 
 
Funder(s) 
FASTMAN Centre of Excellence (PCUK) 
Bristow Funds, Robert G. Bristow, The Manchester Cancer 
Research Centre; 
CRUK Manchester Institute ± Bristow Leadership funds 
Email: robert.bristow@manchester.ac.uk 
Key Collaborators 
Professor Catharine West, PhD, Translational Radiobiology, The 
University of Manchester; 
Email: catharine.west@manchester.ac.uk 
Professor Caroline Dive, PhD, Cancer Research UK Manchester 
Institute, Cancer Biomarker Centre (CBC), The University of 
Manchester; Email: caroline.dive@manchester.ac.uk 
Substance supply / vendor 
(Pimonidazole) 
Mawdsley Brooks & Co Ltd., Pharmaceutical Distributor, 
Company No. 44701, 3 South Langworthy Road, Salford, M50 
2PW, UK; 
Phone: +44 (0)844 736 9016; Email: info@mawdsleys.com 
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TABLE of CONTENTS 
1 ABBREVIATIONS 
8 
2 STUDY SUMMARY 
9 
3 FUNDING 
11 
4 STUDY SCHEME 
12 
5 STUDY INTERVENTIONS 
13 
6 BACKGROUND 
 
14 
6.1 Background 
6.2 Rationale 
6.3 Theoretical Framework 
6.4 Research Hypotheses 
 14 
 15 
 15 
 15 
7 RESEARCH AIMS 
16 
8 STUDY PROCEDURES & METHODOLOGY 
17 
8.1 Study design 
8.2 Study visits and end of study 
8.2.1 
Pimonidazole hydrochloride 
8.3 Patient Benefit and Overall Risk 
8.4 CT-guided Bone Biopsy (Arm 1) 
8.5 TRUS-guided Targeted Transperineal Prostate Biopsy (Arm 1) 
8.6 Radical Prostatectomy (Arm 2) 
8.7 Blood sampling 
8.8 Whole-body MRI (Arm 1) 
8.9 Prostate MRI scans (Arm 2) 
8.10 Tissue processing and storage 
8.11 Hypoxia assessment and analysis of tumour genome and transcriptome 
8.12 Assessment of circulating tumour cells and circulating tumour DNA 
8.13 Storage and use of tissue and blood sample left-over 
17 
 18 
 19 
 20 
 22 
 22 
 23 
 23 
 25 
 25 
 25 
 26 
26 
27 
9 PATIENT SELECTION AND RECRUITMENT 
28 
9.1 Patient Identification 
9.2 Sample Size 
9.3 Patient Selection Criteria 
9.4 Inclusion Criteria 
9.5 Exclusion Criteria 
9.6 Study Payment 
9.7 Informed Consent 
9.8 Ineligible and Non-recruited Patients 
9.9 Study Registration 
 28 
 28 
 29 
 30 
 31 
 32 
 32 
 32 
 33 
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9.10 Withdrawal of Participants 
 33 
 
10 DATA MANAGEMENT 
34 
10.1 Case Report Forms 
10.2 Data Flow 
10.3 Storage of Imaging and Sequencing Data 
 34 
 34 
 34 
11 ADMINISTRATIVE AND REGULATORY DETAILS 
35 
11.1 Study Sponsor 
11.2 Funding Body 
11.3 Peer Review 
11.4 Patient and Public Involvement 
11.5 Ethical Approval 
11.6 Confidentiality 
11.7 Protocol Compliance 
11.8 Trial Monitoring 
11.9 Study Oversight 
11.10 Protocol Amendments 
11.11 Liability and Indemnity 
11.12 Adverse Events and Adverse Reactions 
11.13 End-of-Study Notification 
11.14 Study Summary Report 
11.15 Archiving 
11.16 Publication Policy 
 35 
 35 
 35 
 35 
 36 
 36 
 37 
 37 
 37 
 38 
 38 
 39 
 40 
 40 
 40 
 40 
12 REFERENCES 
41 
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1 ABBREVIATIONS 
AE 
 
 
Adverse event 
APR 
 
 
Annual progress report 
AR 
 
 
Adverse reaction 
CEP 
 
 
Clinical and Experimental Pharmacology Group 
CRUK MI 
 
Cancer Research UK Manchester Institute 
CT 
 
 
Computed tomography 
CTC 
 
 
Circulating tumour cells 
ctDNA  
 
Circulating tumour deoxyribonucleic acid 
CTIMP  
 
Clinical Trial of an Investigational Medicinal Product 
DNA 
 
 
Deoxyribonucleic acid 
ECOG  
 
Eastern central oncology group performance status 
EDTA  
 
Ethylenediaminetetraacetic acid 
EMT 
 
 
epithelial-mesenchymal-transition 
EpCAM  
 
Epithelial cell adhesion molecule 
GCP 
 
 
Good clinical practice 
GLUT1  
 
Glucose Transporter 1 
HRA 
 
 
Health Research Authority 
IPU 
 
 
Interventional Procedures Unit 
MCCBS 
 
Manchester Centre for Cancer Biomarker Sciences 
MCRC  
 
Manchester Cancer Research Centre 
mpMRI  
 
multiparametric magnet resonance imaging 
mRNA  
 
messenger ribonucleic acid 
NAB 
 
 
Nucleic Acid Biomarkers group 
PCa 
 
 
Prostate cancer 
PIMO  
 
Pimonidazole hydrochloride 
PIS 
 
 
Patient information sheet 
PSA 
 
 
Prostate specific antigen 
PTEN  
 
Phosphatase and tensin homolog 
REC 
 
 
Research Ethics Committee 
RNA 
 
 
Ribonucleic acid 
RNAsep 
 
Ribonucleic acid sequencing 
ROI 
 
 
Region of interest 
SAE 
 
 
Serious adverse event 
SOAR  
 
Sponsor oversight activity report 
SOC 
 
 
Standard of care 
SOP 
 
 
Standard operating procedure 
SRC 
 
 
Safety Review Committee 
SUSAR  
 
Suspected Unexpected Serious Adverse Reaction 
TMG 
 
 
Trial Management Group 
TOG 
 
 
Translational Oncogenomics Group 
TRUS  
 
Trans-rectal ultrasound 
USM 
 
 
Urgent Safety Measure 
WES 
 
 
Whole-exome sequencing 
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2 STUDY SUMMARY 
Study Title 
Illuminating the genomic landscape of hypoxia-driven early 
metastatic and localised prostate cancer 
Acronym 
HYPROGEN 
Study Design 
Prospective, non-randomised, exploratory biopsy and 
imaging biomarker study 
Target Patient Population 
Arm 1 - De novo, treatment-naïve metastatic prostate 
cancer 
Arm 2 ± De novo, treatment- naïve localised prostate 
cancer planned for radical prostatectomy 
Planned Sample Size 
Arm 1 ± 30 
Arm 2 - 30 
Planned Study Period 
03 / 09 / 2021 ± 03 / 09/ 2023 
Research Question/Aim(s) 
Assess the differential genomic aberrations and gene 
expressional alterations in hormone-naïve primary PCa and 
paired skeletal metastases. 
Determine whether co-existent intra-prostatic hypoxia, EMT 
and genomic instability is a co-factor in driving early spread.  
Identification of co-existence of hypoxia in primary and 
metastatic tumour sites to identify co-existent hypoxia in 
primary PCa and paired bone metastases. 
Assess the prevalence and origin of CTCs in early, 
aggressive (oligo-) metastatic disease. 
 
Validation of MRI imaging biomarkers for detection of 
hypoxia subregions within primary PCa 
Arm 1 
 
Inclusion criteria 
Male patients aged 18 years and older 
Histologically proven adenocarcinoma of the prostate 
F7 
OR 
Highly suspected metastatic prostate cancer  
36$YDOXHRIQJP/ 
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routine imaging procedures with at least one amenable* to 
biopsy (cohort A) 
OR 
oligometastatic ERQH GLVHDVH  WR   DW URXWLQH ERQH
scan with at least one lesion amenable* to biopsy (cohort B) 
*e.g. safely to biopsy and expectably providing sufficient tissue yield 
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World Health Organisation (WHO) performance status 0 to 
2 with no deterioration over the previous 2 weeks and 
minimum life expectancy of 12 months 
No prior local and/or systemic treatment for localised 
prostate cancer 
Willing to donate cancer tissue samples for research 
purposes (bone metastasis and primary tumour) 
Exclusion criteria 
 
Involvement in the planning and/or conduct of the study 
(applies to staff at the study site) 
Previous enrolment in the HYPROGEN study 
As judged by the investigator, any evidence of severe or 
uncontrolled 
systemic 
disease 
(e.g. 
uncompensated 
respiratory, cardiac, hepatic or renal disease) 
Evidence of any other significant clinical disorder or 
laboratory finding that made it undesirable for the patient to 
participate in the study 
Any investigational agents or study drugs from a previous 
clinical study within 30 days of the first tissue collection 
Prior treatment of localized prostate cancer including 
radiotherapy and/or androgen-deprivation therapy 
Judgment by the investigator that the patient should not 
participate in the study if the patient is unlikely to comply 
with study procedures, restrictions and requirements 
Contra-indications to MRI (incl. pacemakers etc.)  
Bone metastases in difficult to reach areas or areas which 
might be at risk for pathological fracture post biopsy as 
judged by biopsying radiologist / chief investigator 
Increased risk of bleeding as a result of biopsy 
History of bleeding disorders or thrombocytopenia (platelets 
<100/nL) 
Concomitant treatment with anticoagulant therapy, e.g. 
warfarin/low molecular weight heparin or Anti-Xa-inhibitors 
and other NOACs, if temporary cessation medically not 
justifiable  
Current urinary tract infection (UTI) or prostatitis 
Arm 2 
 
Inclusion criteria 
Male patients aged 18 years and older 
cT2-T3 / cN0-N1 / cM0 
Any Group Grade (GG) 2-5: this includes Gleason scores 
3+4, 4+3, 4+4, 4+5, 5+3, 5+4, 5+5. 
Any PSA 
 
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3 FUNDING 
FUNDER(S) 
FINANCIAL AND NON FINANCIALSUPPORT GIVEN 
FASTMAN Centre of 
Excellence (PCUK) ,  
CRUK MI ± Bristow Funds 
 
Financial coverage for whole study conduct 
Histologically proven adenocarcinoma of the prostate 
Undergoing radical prostatectomy as primary treatment for 
localised prostate cancer 
World Health Organisation (WHO) performance status 0 to 
2 with no deterioration over the previous 2 weeks and 
minimum life expectancy of 12 months 
No prior local and/or systemic treatment for localised 
prostate cancer 
Willing to donate cancer tissue samples for research 
purposes (any metastasis and primary tumour) 
Exclusion criteria 
 
Involvement in the planning and/or conduct of the study 
(applies to staff at the study site) 
 
As judged by the investigator, any evidence of severe or 
uncontrolled 
systemic 
disease 
(e.g. 
uncompensated 
respiratory, cardiac, hepatic or renal disease) 
 
Any investigational agents or study drugs from a previous 
clinical study within 30 days of the first tissue collection 
 
Prior treatment of localized prostate cancer including 
radiotherapy and/or androgen-deprivation therapy 
 
Judgment by the investigator that the patient should not 
participate in the study if the patient is unlikely to comply 
with study procedures, restrictions and requirements 
 
Contra-indications to MRI (incl. pacemakers etc.)  
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4 STUDY SCHEMEArm 1
*SOC, standard of care: Androgen deprivation ± Abiraterone Acetate or Docetaxel or Enzalutamide ± radiotherapy to the 
prostate; check study availability, i.e. STAMPEDE, etc. 
Arm 2 
 
 
5 STUDY INTERVENTIONS 
Arm 1 
Visit 
Consent 
Biopsy 
Imaging 
Follow-up 
History taking 
ź 
 
 
 
Physical exam 
ź 
 
 
ź 
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TRUS 
 
ź 
 
 
- 
Prostate biopsy 
 
ź 
 
 
CT scan ROI 
 
ź 
 
 
- 
Bone biopsy 
 
ź 
 
 
wb MRI 
 
 
ź 
 
Blood sampling (routine) 
ź 
 
 
 
Blood sampling (research) 
ź 
ź 
 
ź (patients 
11-30 only) 
CT, computed tomography; wb MRI, whole body magnet-resonance imaging; ROI, region of interest; TRUS, transrectal 
ultrasound 
 
Arm 2 
Visit 
Consent 
1st MRI 
scan  
2nd MRI 
scan 
Procedure 
History taking (standard of care) 
ź 
 
 
 
Physical exam (standard of care) 
Diagnostic and staging tests 
(Standard of care) 
ź 
 
 
 
Radical Prostatectomy (standard 
of care) 
 
 
 
ź 
MRI scan 
 
ź 
ź 
 
Blood sampling (standard of 
care) 
ź 
 
 
 
Blood sampling (research) 
ź 
 
 
 
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6 BACKGROUND 
6.1 Background 
Metastatic prostate cancer  
Prostate cancer is the most common malignancy in men and the second commonest 
cause of cancer-related deaths in Western countries. Around 47,700 patients will be 
diagnosed with prostate cancer (PCa) in the UK each year [1]. About 15% present with 
primary M1 disease, which remains incurable despite remarkable improvements of 
treatment over the last decade. Interestingly, about 60% of M1 patients show solely 
metastasis to the bone sparing lymph nodes and any other organs. However, there is a 
paucity of data explaining both, early metastatic spread and the preference of prostate 
cancers to metastasise to the bones.  
Oligometastatic disease 
Oligometastatic solid tumours refers to a stage with a limited number of metastases and 
is perceived to be a distinct subtype of malignancy where more aggressive or at least 
more effective treatment holds promise to sustainably eradicate all lesions and thus to 
be curative [2]. For PCa, oligometastatic disease has not yet been adopted as an own 
subtype of diseDVH EXW LW FDQ EH GHILQHG DV WKH SUHVHQFH RI   QRQ-locoregional 
metastases. Recently, the STAMPEDE study reported a remarkable survival 
improvement by the addition of radiotherapy of the prostate to androgen-deprivation as 
standard of care in patients with a maximum of 4 bone metastases as identified by 
routine bone imaging, which is why we decided to apply a cut-RIIRIERQHPHWDVWDVHV
on routine bone scintigraphy to define a PCa as being oligometastatic [3]. Consequently, 
novel targeted, biomarker-driven treatment approaches may ultimately help to improve 
treatment success, particularly in oligometastatic PCa.  
Tumour hypoxia 
The tumour microenvironment has a strong influence on tumour growth and progression, 
which renders it a potential target for novel treatment approaches. For instance, it is 
characterised by dynamic gradients of oxygen diffusion and consumption leading to sub-
regions of hypoxia in about half of all solid tumours. Tumour adaptation to imbalanced 
oxygen supply and demand is associated with elevated genomic instability, resistance to 
chemotherapy and radiotherapy, immune dampening, altered metabolism, cell death 
resistance, development of tumour stem cell protective niches, increased mobility and 
invasiveness though epithelial-mesenchymal-transition (EMT) of tumour cells and 
increased proclivity for distant metastasis, such as bone metastases, among others [4-
6]. Consequently, cancer patients with hypoxic tumours, including PCa, have a dismal 
prognosis irrespective of the applied treatment modality [7]. Given its central role in 
tumour aggressiveness and progression and treatment resistance, tumour hypoxia might 
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well be considered the best validated target that has yet to be successfully exploited in 
oncology [8]. 
 
6.2 Rationale 
Ongoing Phase III studies are testing whether oligometastatic disease can be eradicated 
with local ablation in addition to best systemic care; but a mechanistic basis for further 
personalised intensified treatment in the systemic arms, e.g. by adding anti-hypoxia 
compounds for the most hypoxic tumours, is lacking. As hypoxia is scarce in normal, 
vital tissues, targeting this characteristic of aggressive PCa will most likely primarily kill 
cancer cells and augment the therapeutic ratio for patients with both, oligometastatic and 
widespread metastatic disease. 
 
6.3 Theoretical Framework 
As described in the background section, tumour hypoxia is a driver of tumour 
aggressiveness conferring a poor prognosis. Hypoxia can select for cancer cells that are 
apoptosis-deficient, contain TP53 mutations, and have increased genomic instability 
leading to a mutator phenotype [7]. Importantly, the Bristow lab previously reported that 
the co-presence of tumour hypoxia (based on mRNA signatures or needle electrode 
measurements) and genomic instability synergistically portend rapid relapse after 
primary treatment for PCa, supporting the concept that a hostile tumour 
microenvironment may select for or drive adaptation of a distinctive genomic profile and 
rapid failure due to occult metastases [9]. In line with this, a study from the West group 
showed that a prostate-specific hypoxia-associated 28-gene RNA expression signature 
portends poor prognosis [10]. Therefore, we aim to comprehensively assess the impact 
of a hypoxic primary tumour on the oxygenation of its bone metastases and the resulting 
genomic and gene expressional aberrations supporting the development of early 
metastatic spread to the bone. 
 
6.4 Research Hypotheses 
1. Hypoxia drives genomic instability in primary prostate cancers.  
2. Co-existent intra-prostatic hypoxia and genomic instability is a co-factor in driving 
early metastatic spread to the bone. 
3. Hypoxia drives migration and invasion increasing the prevalence of circulating 
tumour cells (CTCs) disseminating from the primary tumour to form oligo 
metastases 
4. Hypoxia is present in skeletal metastases themselves. 
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5. Primary prostate cancer and early bone metastases have a distinct genomic and 
transcriptomic profile related to hypoxia. 
6. CTC genomes more strongly correlate with hypoxic regions of primary tumour. 
7. MRI hypoxia sequences are able to detect hypoxic sub-regions within prostate 
cancer. 
 
7 RESEARCH AIMS 
Assess the differential genomic aberrations and gene expressional alterations 
in hormone-naïve primary PCa and paired skeletal metastases. 
Whole exome sequencing (WES) analysis will be performed and the results of the paired 
samples of the study patients will be compared in silico to ~500 patients with pT1-3 
N0M0 disease from the CPC-GENE bioinformatics portal as a control set [11]. Moreover, 
to assess the gene expression profile of primary tumour and metastases, the whole RNA 
will be sequenced (RNAseq). 
Determine whether co-existent intra-prostatic hypoxia, EMT and genomic 
instability is a co-factor in driving early spread  
Hypoxic areas of primary tumour identified through either pimonidazole or other hypoxia 
labelling method such as GLUT1 immunohistochemical staining (if available, otherwise 
archival FFPE tissue from primary diagnostic biopsy) and metastatic tumour tissue will 
be micro-dissected and analysed by WES, RNAseq, and a targeted mRNA analysis to 
detect the Manchester 28-gene expression signature predicting for tumour hypoxia. Data 
pertaining 
to 
the 
co-occurrence 
in 
regions 
identified 
as 
hypoxic 
using 
immunohistochemical stains (such as pimonidazole or GLUT1), Copy Number 
Alterations, Single Nucleotide Variants and whole RNA expression profiles as well as the 
Manchester hypoxia 28-gene RNA signature will be compared.  
Identification of co-existence of hypoxia in primary and metastatic tumour 
sites 
Results of immunohistochemically identified hypoxic regions (through PIMO-staining or 
alternate staining method such as GLUT1) and the 28-gene RNA expression of the 
samples will be compared for each patient to identify co-existent hypoxia in primary PCa 
and paired bone metastases.  
Assess the prevalence and origin of CTCs in early, aggressive (oligo-) 
metastatic disease 
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To increase sensitivity of detection marker-independent platforms will be used. CTCs will 
be isolated and molecularly profiled to determine whether they originate from primary 
tumour and/or from metastases. Moreover, CTC genomes will be mapped to 
primary/metastatic tumour regions identified as having high or low levels of hypoxia 
(through pimonidazole or other staining method) to determine if hypoxia (and hypoxia 
associated EMT) drives tumour cell dissemination. 
 
8 STUDY PROCEDURES & METHODOLOGY 
8.1 Study Design 
This is a prospective, non-randomised, single-centre, exploratory biomarker study. In 
Arm 1 this involves targeted tumour tissue sampling in men with either newly diagnosed, 
untreated metastatic prostate cancerinvolving patients with either high (cohort A; 
widespread metastatic disease) or low, oligometastatic (cohort B; ERQHPHWDVWDVHV 
burden of disease. In Arm 2, targeted tumour tissue samples derived from prostate 
cancers biobanked as per standard MCRC biobanking protocol in 30 men undergoing a 
radical prostatectomy as part of their standard care. 
 
In Arm 1, to minimize the interventional burden for the patients, patients will be 
amenable to have a combined diagnostic and research biopsy of the prostate in one 
session after routine imaging for diagnostic work-up comprising of a CT scan of 
abdomen and pelvis, bone scan and multi-parameteric MRI of the prostate. Patients, 
who already had a diagnostic TRUS biopsy will be asked to provide research tissue 
samples during a second TRUS biopsy as part of the HYPROGEN study interventions. 
Patients declining a second prostate biopsy will be eligible and will undergo the bone 
biopsy only. Patient identification will be done through the respective multidisciplinary 
tumour (MDT) boards and patient referral pathways at Salford Royal, Oldham Royal and 
Wythenshawe Hospital as further outlined in section 9.1. 
To yield highest possible biopsy accuracy and tissue amount and maximum procedure 
safety, prostate biopsy will be a TRUS-guided targeted transperineal biopsy and bone 
biopsy will be CT-guided using an automated drill. Moreover, to identify the imaging 
modality with the highest accuracy in detecting bone-metastatic prostate cancer at first 
presentation, an additional whole-body MRI will be applied to patients on the study. 
Study participation will not affect the routine care for this stage of disease, which will 
start immediately after the biopsy samples have been acquired. 
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In Arm 2 tumour tissue samples will be acquired from prostatectomy specimens that 
patients are undergoing as part of there routine standard of care.  
 
8.2 Study Visits and End of Study 
Arm 1 Once a patient has been identified at a referring urology centre at Oldham, 
Salford or Wythenshawe to meet the HYPROGEN Arm 1 study inclusion criteria, he will 
be informed about this trial by the local care team and provided with the participant 
information leaflet. The patient will be asked for permission to be contacted by the 
HYPROGEN study staff at The Christie NHS Foundation Trust to evaluate his interest in 
study participation. If a patient is willing to participate, he will then be invited to an 
outpatient research clinic to recapitulate potential risks and benefits of study participation 
(consent visit).  
If a patient agrees to participate, the informed consent form will be signed and the 
optional non-IMP pimonidazole will be prescribed by the consenting research team 
member. The hypoxia marker agent pimonidazole will be dispensed by The Christie 
Research Pharmacy, and the participant will be asked to take it at home the day before 
the biopsy visit.  
Within a further 7 working days from consent to the study, study participants will receive 
a CT-guided bone biopsy and a TRUS-guided transperineal biopsy of their prostate 
gland as an outpatient. During the following 10-15 working days, patients will undergo a 
whole-body MRI scan at the Radiology Department of The Christie.  
If the patient refuses the pimonidazole, forgets to take it, or if it is not available, the 
patient can still participate in the study and their samples will be stained for hypoxia 
post-biopsy. 
After the biopsy, patients will be referred back to their consultant urologists at Oldham, 
Salford or Wythenshawe for standard routine care or discussion of additional treatment 
options, i.e. inclusion in the STAMPEDE study, which almost all patients eligible for the 
HYPROGEN study will be eligible for. The treatment as per standard of care in 
accordance with the recommendation of the MDT discussion will commence on the day 
after the biopsy visit. 
Within the 3-6 months following the biopsy, the patients will be invited back to the 
Christie to provide a follow-up research blood sample. The study ends after the follow-up 
visit of the last participant. 
 
Arm 2  
In patients identified as eligible for Arm 2 of the Hyprogen study, the study will be 
discussed with eligible patients by their referring consultant and provided with the 
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participant information leaflet. The patient will be asked for permission to be contacted 
by the PROXIES study staff at The Christie NHS Foundation Trust to evaluate his 
interest in study participation. If a patient is willing to participate, he will then be invited to 
The Christie outpatient research clinic to recapitulate potential risks and benefits of study 
participation (consent visit). If a patient agrees to participate, the informed consent form 
will be signed and the non-IMP pimonidazole will be prescribed.  
The hypoxia marker agent pimonidazole will be dispensed by The Christie Research 
Pharmacy, and the participant will be asked to take it at home the day before planned 
radical prostatectomy.  
The study ends after the last patient has undergone a radical prostatectomy.  
 
 
8.2.1 Pimonidazole Hydrochloride 
For the purposes of this study patients will be asked to ingest an oral formulation of 
pimonidazole hydrochloride (HCl) (Oral HypoxyprobeTM-1). Pimonidazole HCl is a 
marker for hypoxia in tumour tissue when ingested as an encapsulated solid. Following 
oral administration, pimonidazole distributes throughout the body where it covalently 
binds to normal and tumour tissues that have regions of low oxygen concentrations (pO2 
RIPP+JDWoC). The tissue binding can be visualised by immunohistochemistry / 
light microscopy. Pimonidazole HCl has been issued by the US Food and Drug 
Administration as Investigational New Drug status as a diagnostic for human tissue 
hypoxia and a number of studies in the US, Canada and Europe use encapsulated 
pimonidazole HCl to study of human tumour hypoxia in vivo. Pimonidazole HCl is given 
orally as capsules at a dosage of 0.5 g/m2 body surface area. For oral administration, 
pimonidazole HCl is supplied in the form of capsules containing 200 mg (coloured white) 
and 300 mg (coloured orange brown) of the substance, admixed with lactose excipient in 
the case of 200 mg capsules. The colouring is supposed to prevent errors in the choice 
of capsules. A clinical investigation begins with a calculation of the total amount of 
pimonidazole HCl needed to achieve a dosage of 0.5 g/m2. For choosing the correct 
number of capsules, the total amount of pimonidazole HCl in milligrams is rounded to the 
nearest whole number according to WKHLQYHVWLJDWRU¶VEURFKXUH (see following table). 
Total amount of 
pimonidazole HCl 
200 
mg 
capsules 
300 
mg 
capsules 
Total 
No. 
of 
capsules 
600 mg 
0 
2 
2 
700 mg 
2 
1 
3 
800 mg 
1 
2 
3 
900 mg 
0 
3 
3 
1000 mg 
2 
2 
4 
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1100 mg 
1 
3 
4 
1200 mg 
0 
4 
4 
 
The dose limiting toxicity for pimonidazole hydrochloride in humans was identified, when 
pimonidazole was evaluated as a radiosensitising agent for solid malignancies. It was 
central nervous system toxicity [11, 12]. Importantly, oral administration of 0.5 g/m2 has 
been well tolerated without any signs of toxicity. 
Following ingestion, pimonidazole HCl is distributed to all tissues in the body including 
the brain but forms adducts with proteins only in those cells that have oxygen 
concentrations, e.g. a pO2 of less than 10 mm Hg at 37°C. In addition to tumours, a 
number of normal tissues such as liver, kidney and skin possess cells at, or below, a 
pO2 of 10 mmHg. These normal tissues will also bind pimonidazole. Pimonidazole HCl 
is primarily eliminated via urinary excretion mostly within 72 hours of ingestion, a small 
amount appears in the faeces or gets eliminated by tissue metabolism. 
The capsules are to be taken within 8-16 hours (optimal timepoint 12 hours) before 
the planned first biopsy within Arm 1 and before radical prostatectomy for 
patients in Arm 2. If the patient refuses the pimonidazole, forgets to take it, or if it is not 
available, the patient can still participate in the study and their samples will be stained for 
hypoxia post-biopsy. 
 
8.3 Patient Benefit and Overall Risk 
This is a prospective, translational, exploratory biomarker study, which is why there will 
be no direct benefit derived from the data obtained for the individual PCa patient 
participating in this study. However, this study may ideally lead to the identification of 
tumour hypoxia and its related aberrations as a therapeutic target in (oligo-)metastatic 
PCa in the near future. Results of the HYPROGEN study will not affect treatment 
according to the current standard of care including - for patients in Arm 1 - androgen 
deprivation with or without more intensified treatment including docetaxel, abiraterone 
acetate plus prednisone and/or irradiation of the prostate. 
Within Arm 1 Patients who are enrolled onto this study will need to make four additional 
outpatient visits to the hospital for the duration of the study (consent, biopsy, imaging, 
and follow up visit).  
Patients entering this study will undergo additional imaging with a whole-body MRI as 
well as additional biopsies of tissues affected by their cancer at two body areas as well 
as additional blood sampling for research purposes. The tissue biopsy sites include the 
prostatic gland and the bones. There may be side effects associated with these biopsies.  
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The additional whole-body MRI scan will help to further define the sensitivity of routine 
bone scanning and MRI scanning to detect a metastatic state. 
Biopsies of the prostate are part of normal regular care performed on all patients with de 
novo metastatic PCa. As for routine diagnostic purposes, prostate biopsies will be done 
though the perineum guided by trans-rectal ultrasound (TRUS) targeting lesions, which 
may have already been identified in the pre-study diagnostic multiparametric MR scan of 
the prostate (if available). Preferably, routine diagnostics and research sampling will be 
undertaken during the same biopsy procedure. However, if a diagnostic TRUS biopsy of 
the prostate has been performed before, the research biopsy will require an additional 
intervention surplus the bone biopsies and the blood sampling, which are not part of 
routine diagnostics and thus cannot be seen as standard of care. If a second prostate 
biopsy solely for research purposes is denied by the patient, only a bone biopsy and a 
blood sample will be taken. Importantly, the techniques of image-guided biopsy taking 
are routinely done for the diagnosis and management of patients with different types of 
cancers and thus safely applicable. As part of the HYPROGEN study, the bone biopsies 
will be used to histologically confirm the diagnosis of metastatic disease in a 
radiographically bone metastatic stage. 
Most side effects associated with the medical procedures in this study are expected to 
be mild to moderate and to disappear after a short time. The main risks related to the 
HYPROGEN study interventions comprise the following: 
 
Pain or Discomfort 
Biopsies involve needle tests of deeper tissues and can cause pain or discomfort both at 
the time of the biopsy and also for up to several days after the biopsy. Local 
anaesthetics injection will be given for both the prostate biopsies as well as the bone 
biopsies to numb the respective site as good as possible to reduce the possible 
discomfort. The local anaesthetic injection itself may cause some temporary pain or 
burning sensation until the onset of numbness, and as the anaesthetics wear off pain 
may re-appear. Simple systemic analgesics such as paracetamol may be advised if pain 
occurs as a result of the biopsies. 
Bleeding or Bruising 
Any needle test can be associated with bleeding or bruising. Blood clotting will be 
assessed during the screening process and patients will not proceed to the biopsy visit if 
they appear to be at higher bleeding risk than normal. Anticoagulant treatment will be 
halted if medically justifiable. Biopsies close to higher risk areas such as large blood 
vessels will be avoided. However, despite these precautions, rarely the bleeding from 
biopsies can cause significant blood loss or risk to life. 
Infections  
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Patients are vulnerable to infection while having a biopsy or blood sample taken. Minor 
infections can become life-threatening. Symptoms of infection include fever, shivering, 
sweats, sore throat, diarrhoea, dysuria, haematuria, cough or breathlessness. Patients 
will be asked to measure the core body temperature on the days after the biopsy visit 
and to report any febrile temperatures or signs of infection immediately to the study staff 
or via The Christie Hotline under 0161 446 3658, which is open 24/7. 
All patients undergoing a transperineal prostate biopsy will receive antibiotic prophylaxis 
as per local policy for prophylactic antibiotic treatment for prostate biopsies. 
 
Arm 2  
This is a prospective, translational, exploratory biomarker study, and it is unlikely to 
directly benefit the participants of this study. However, the results of this study may 
provide benefit for prostate cancer patients in the future. 
Participants will take an oral pimonidazole tablet prior to their radical prostatectomy as 
part of the study. Oral pimonidazole at a dose of 0.5 g/m2 has been well tolerated without 
any signs of toxicity. 
Participants in Arm 2 agreeing to additional imaging will undergo 2 additional MRI scans 
during which they will receive intravenous gadolinium contrast and be asked to breath 
100% oxygen through an oxygen mask. 
 
 
 
8.4 CT-guided Bone Biopsy (Arm 1) 
A CT-guided biopsy of a bone metastasis that is deemed to be easy to biopsy and in an 
area without major risk for pathological fracture or bleeding will be taken during the 
biopsy visit. Patients will receive routine local anaesthetic of the region to be biopsied 
followed by thorough disinfection of the biopsy site with antiseptic wipes. Patients will be 
asked to fast on the day of the procedure and to have an intravenous cannula inserted to 
allow the use of medication causing minimal sedation (for example midazolam and/or 
fentanyl) during the procedure if required to alleviate discomfort or pain. For biopsy 
sampling the Arrow® OnControl® Powered Bone Access System (Teleflex, USA) will be 
used. In patients with greater than one bone metastasis assessed as amenable to bone 
biopsy, biopsies will be obtained from a maximum of two sites of bone metastases. A 
maximum of three specimens from each biopsy site will be taken. Samples will be 
examined on site by a histopathologist to warrant collection of high-quality material and 
advise the sampling as needed. Maximum duration of procedure will be 45-60 min. 
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Following the bone biopsy, the patient will be observed in the Radiology Department to 
be assessed for any excessive bleeding before transferring.  
 
8.5 TRUS-guided Targeted Transperineal Prostate Biopsy (Arm 1) 
Transperineal Prostate Biopsy will be performed following standard clinical practice of 
local department. This will include pre-operative oral analgesia and prophylactic 
antibiotic treatment according to local hospital policy for transperineal prostate biopsies.  
Patients will be placed in lithotomy position andthe scrotum will be carefully taped out of 
the way. As per local hospital practice the procedure may - at the discretion of the 
performing surgeon - involve application of topical ointment (ie 2% diltiazem) to relax the 
anal sphincter, insertion of a transurethral catheter for the procedure and/or the use of a 
local lidocaine-containing gel (Instillagel) inserted into the patients back passage and 
applied to the anus to alleviate discomfort related to the ultrasound probe. The perineal 
skin will be cleaned with an antiseptic solution. An ultrasound probe is then passed into 
the back passage, which will remain there for the duration of the procedure to visualise 
the prostatic gland to guide perineal sampling. In order to numb the perineum and 
prostate as much as possible, a local anaesthetic will then be injected underneath the 
skin and into the deeper tissue layers guided by ultrasound. Systematic and targeted 
core biopsies from the prostate will be acquired.In addition to core biopsies required for 
standard of care diagnostic purpose, lesions seen on a mpMRI (if available) will be 
targeted to allow a minimum of 4 core biopsies from any detected lesion as well as at 
least 2 core biopsies from uninvolved prostate tissue.Otherwise cognitive biopsies 
guided by ultrasound will be obtained. If a second prostate lesion is detected, core 
biopsies will also be acquired from this lesion. In total, in addition to any core biopsies 
required as standard of care a maximum number of 8 to 12 additional prostate core 
biopsies will be taken for research purposes.  Maximum duration of procedure will be 60 
minutes. 
 
8.6 Radical Prostatectomy (Arm 2) 
Radical Prostatectomy will be performed according to standard of care robotic approach 
used at The Christie Hospital and as relayed to the patient by the attending urologic 
surgeon. The side effects of the surgery are the ones reported in the literature and the 
latest participant information leaflet provided prior patient consent (e.g. risk of erection 
disfunction, incontinence, etc.). 
The current study will not imply any change of the current surgical procedure as 
standard of care conducted routinely at the Christie Hospital. It will not increase the 
incidence of known potential co-morbidities already described in general for a radical 
prostatectomy nor instructions to the patient by the attending urologic surgeon. 
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8.7 Blood Sampling 
For patients in Arm 1 - A blood sample for research purposes will be withdrawn at the 
each of the following visits: 
(i) 
Baseline visit (pre- pimonidazole) 
x 
2 x 10mL Streck cell-free DNA blood collection tubes® for circulating tumour 
cell (CTC) collection and circulating tumour DNA (ctDNA) extraction.  
x 
1-2 x 3.5mL blood samples collected into EDTA tubes for germ line DNA 
extraction and processing to PBMC for banking and future profiling of 
immune cell populations 
x 
1 x 10mL serum tubes for future, biobank related research projects, ie 
lipidomics, metabolics and microRNA analysis 
 
(ii) 
Biopsy visit (pre-biopsy) 
x 
2 x 10mL Streck cell-free DNA blood collection tubes® for circulating tumour 
cell (CTC) collection and circulating tumour DNA (ctDNA) extraction.  
x 
1 x 10mL serum tubes for future, biobank-related research projects, ie 
lipidomics, metabolics and microRNA analysis 
 
(iii) 
Follow up visit (within 6 months post-treatment, for patients 11-30)  
x 
2 x 10mL Streck cell-free DNA blood collection tubes® for circulating tumour 
cell (CTC) collection and circulating tumour DNA (ctDNA) extraction.  
x 
1-2 x 3.5mL blood collected into EDTA tubes for germline DNA extraction and 
processing to PBMC for banking and future analysis of immune cell 
populations 
x 
1 x 10mL serum tubes for future, biobank related research projects, ie 
lipidomics, metabolics and microRNA analysis 
 
For Patients in Arm 2 
 
A blood sample (maximum 20ml) will be taken for standard of care blood tests prior to 
prostatectomy including Full Blood Count, Renal Function and PSA. At the same time 
these standard of care bloods are taken, additional bloods - a maximum of 30ml ± will be 
taken for research purposes as required for the following downstream analysis: 
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x 
Germ line DNA extraction and optional processing to PBMC for banking and 
future profiling of immune cell populations 
x 
Biobank related research projects, ie lipidomics, metabolics and microRNA 
analysis 
 
 
8.8 Whole-body MRI (Arm 1) 
For patients within Arm 1, Whole-body MR imaging (wbMRI) will be performed once, 
before or after the biopsy study visit, depending on available examination slots in the 
Department of Radiology at The Christie NHS Foundation Trust. wbMRI images will 
allow comparison of the numbers of bone metastases detected by routine bone scan 
and wbMRI for sensitivity assessment of both techniques for oligometastatic disease. 
MR Sequences acquired following Christie Hospital Whole Body MRI Image Acquisition 
Guidelines. The wbMRI scan is estimated to last around 70 minutes.  
vist 
8.9 Prostate MRI scans (Arm 2) 
Patients within Arm 2 will be offered the option to undergo additional MR imaging of the 
pelvis in addition to any standard of care imaging acquired. In patients who agree to 
undergo additional scans, MRI scans will be performed on 2 occasions prior to the 
radical prostatectomy. MRI scans will be acquired on either the MR sim diagnostic 
scanner, on the MR Linac scanner or on both within The Christie NHS Foundation Trust.  
The MR sequences obtained will include sequences for oxygen-enhanced MR, IVIM, 
DCE and BOLD and aim to detect hypoxic sub-regions of tumour that can be validated 
against pimonidazole staining regions following prostatectomy. Patients will be asked to 
will breathe 21% followed by 100% oxygen gas through an oxygen mask during the 
scan. They will receive a gadolinium-based contrast agent through an intravenous 
cannula. Scan time will not exceed 60 minutes per scan. Any patients not agreeing to 
undergo additional MRI scans are still able to be enrolled into Arm 2 of the trial.  
 
8.10 Tissue Processing and Storage 
The PrestoCHILL freezing device (Milestone Medical, Menarini Diagnostics, UK) will be 
used in order to access tumour content and select appropriate material for obtaining the 
highest quality DNA and RNA, retaining also an appropriate amount for FFPE evaluation 
and studies. Samples will be collected and stored at the Manchester Cancer Research 
Centre Biobank. Serum samples will be centrifuged and frozen at -20°C at the MCRC 
biobank. The other blood samples will be taken to the biobank at room temperature, 
Streck tubes and EDTA tubes will then be send to the CRUK Manchester Institute 
Cancer Biomarker Centre (CBC) (Prof Caroline Dive). 
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Prostate specimens will be routinely sectioned with a cryotome and sections for 
diagnostic routine H/E histopathology as well as additional sections for pimonidazole or 
alternative hypoxia staining method (such as GLUT1 immunohistochemical staining) and 
nucleic acid extraction for subsequent genomic/transcriptomic analysis cut. Bone 
specimens will be similarly frozen and analysed but cut on the cryotome using special 
cryo tungsten carbide blades. Tissue cryo-preservation, sectioning and processing will 
be carried out by the Histopathology Department at The Christie NHS Foundation Trust 
and Cancer Research UK Manchester Institute (CRUK MI). 
 
8.11 Hypoxia Assessment and Analysis of Tumour Genome and Transcriptome 
Fresh-frozen tumour samples will be analysed by both conventional histopathology and 
comprehensive assessment of the tumour genome and functional gene expression 
profile. 4-5µm sections will be taken for immunohistochemistry, 10 µm sections will be 
taken for nucleic acid extraction. A consultant histopathologist (Dr Pedro Oliveira, The 
Christie NHS Foundation Trust) will (i) confirm the diagnosis of prostate cancer and will 
allow for proper diagnostic work-up including grading according to the Gleason score 
(H/E staining) and (ii) allow assessment of the presence of intratumoural hypoxia as 
detected by immunohistochemical staining for pimonidazole. If the patient did not receive 
pimonidazole pre-biopsy then an alternative immunohistochemical stain such as GLUT1 
will identify hypoxic areas. Hypoxic areas will be marked to guide microdissection for 
subsequent nucleic acid extraction from the respective slides. 
Further analysis of the tumour DNA, RNA and  functional transcriptome will include 
whole exome sequencing (WES), which may be carried out by a third party as 
appropriate, with all necessary agreements in place.   
 
 
8.12 Assessment of circulating tumour cells (CTCs) and circulating tumour DNA 
(ctDNA) 
In collaboration with the CRUK Manchester Institute Cancer Biomarker Centre (CBC) 
(Prof Caroline Dive) blood samples will be used to assess the prevalence and origin of 
CTCs and ctDNA. Previous studies have demonstrated that the presence of 5 or more 
CTCs at baseline, as determined using the CellSearch platform, are a prognostic factor 
for overall survival in metastatic prostate cancer [14, 15]. Although the CellSearch assay 
is the only FDA-approved method for CTC detection in prostate cancer, the fact that the 
system relies on CTCs expressing EpCAM antigen for capture means that CellSearch 
compares unfavourably with marker-independent platforms for CTC capture in this 
disease [16]. The CRUK MI CBC have extensive experience of CTC analysis in a variety 
of clinical settings, and have developed a number of platforms and methods for CTC 
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capture and analysis. In addition to the CellSearch platform, in routine use in the CBC 
are microfluidics-based, marker independent CTC enrichment systems (Parsortix, 
ClearCellFX), and also slide-EDVHG ³QR FHOO LV ORVW´ FDSWXUH V\VWHPV 5DUH&\WH +LJK-
Definition Single Cell Analysis, HD-SCA). These marker-independent platforms are 
amenable to the study of CTCs in prostate cancer. For this study  CTCs will be enriched 
using the Parosortix platform and  enumerated and characterised using bespoke assays 
developed by CBC. Characterisation will include staining of captured cells for known 
CTC and exclusion markers, plus if required bespoke staining assays can be developed 
for markers relevant to prostate cancer, e.g. AR, PTEN. Additionally, single CTCs can be 
isolated for genomic analysis to complement analysis of the tumour genome, and 
analysis of ctDNA. CTC genomes will then be compared to primary and metastatic 
lesions to determine their origin(s). Moreover, it will be assessed if CTC genomes are 
enriched for hypoxic regions (based on pimonidazole labelling or an alternative hypoxia 
biomarker such as GLUT1). 
Profiling of ctDNA is a key aspect of the work of the Nucleic Acid Biomarkers (NAB) 
team, and which complements the CTC analysis outlined above. The NAB team has 
developed methods for the isolation of ctDNA from patient samples, and also analytical 
approaches including gene copy number variation (CNV), targeted sequencing using 
droplet-digital PCR and selected gene panels, and discovery approaches such as whole 
exome sequencing (WES). There are a number of examples where analysis of ctDNA 
from patient blood samples has confirmed and extended detections of mutations in 
primary tumours and metastases in prostate cancer (e.g. [17, 18]). Analysis of patient 
ctDNA is particularly amenable to the stated aims of this study, specifically assessing the 
genomic aberrations in hormone-naïve primary prostate cancer, and determining 
genomic instability driving early spread. Certain genomic aberrations have been 
documented as prognostic in metastatic prostate cancer, including androgen receptor 
point mutations and splice variants [19-21], PTEN loss which is associated with 
decreased AR activity [22], and mutations in DNA repair genes [23]. A panel of these 
genes for targeted sequence analysis based on ctDNA will be applied, consistent with 
the biopsy-driven analysis described earlier. 
 
8.13 Storage and use of tissue and blood sample left-overs 
Patients participating in this study also consent that any unused samples at the end of 
this study will be gifted to the Manchester Cancer Research Centre (MCRC) Biobank for 
use in future research projects. The MCRC Biobank makes samples available to all 
kinds of researchers, including pharmaceutical companies for all kinds of cancer 
research and genetic studies. Access to Biobank samples is strictly regulated and the 
samples will only ever be released for high quality cancer research projects approved by 
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the MCRC Biobank Access Committee. Researchers using samples will only ever 
receive anonymised data and personal identifiable information will be kept strictly 
confidential.  
 
9. PATIENT SELECTION AND RECRUITMENT 
9.1 Patient Identification 
Patients eligible for study inclusion will be identified from their medical records after 
routine staging procedures for newly diagnosed prostate cancer during the respective 
multidisciplinary team meeting (MDTs) or patient referral pathway and then be informed 
of this study by their local clinical care team within the respective urology clinics at the 
University Hospital of South Manchester NHS Foundation Trust in Wythenshawe 
(Urology consultant: Prof V Ramani), The Royal Oldham Hospital (Urology consultant: 
Mr J Oates), and The Salford Royal NHS Foundation Trust (Urology consultants: Prof N 
Clarke, Mr Satish Maddineni).  
The local clinical care team provides eligible patients with the participant information 
leaflet and requests the patient to allow the study staff at The Christie NHS Foundation 
Trust to contact the patient to evaluate his interest in study participation. If a patient is 
willing to participate in the HYPROGEN study an appointment at The Christie outpatient 
UHVHDUFKFOLQLFIRUDQRWKHUGHWDLOHGUHFDSLWXODWLRQRIWKHVWXGLHV¶aims and potential risks 
and benefits of study participation will be scheduled for within the next 5 working days 
(consent visit). Patients that are either not suitable for inclusion or whom do not wish to 
participate will continue along the routine care pathway. 
 
9.2 Sample Size 
A total number of 60 patients will be recruited into this pilot study, 30 patients with 
metastatic disease in Arm 1 and 30 patients with localised prostate cancer in Arm 2. 
Within Arm 1, 15 patients with a high disease burden identified as a number of bone 
PHWDVWDVHVwith or without visceral metastases (cohort A) and 15 with a low burden 
RIERQHPHWDVWDVHVGHILQHGDVERQHPHWDVWDVHV and no signs of visceral disease 
(cohort B) detected on routine bone scan imaging. As this is a feasibility study, no power 
calculation was applied for patient number estimation. 
 
 
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9.3 Patient Selection Criteria 
All patients must meet all of the inclusion criteria and none of the exclusion criteria for 
this study at the time of registration. Under no circumstances can there be exceptions to 
this rule. 
 
 
 
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9.4 Inclusion Criteria 
Arm 1 
 
No 
Inclusion Criteria 
Patients are eligible only if they meet all of the following criteria 
1 
Male patients aged 18 years and older 
2 
+LVWRORJLFDOO\SURYHQDGHQRFDUFLQRPDRIWKHSURVWDWHF7 
OR 
Highly suspected metastatic prostate cancer  
3 
PSA YDOXHRIQJP/ 
4 
0XOWLSOHOHVLRQVVXVSLFLRXVRIPHWDVWDWLFVSUHDGRQURXWLQHLPDJLQJSURFHGXUHVZLWK
at least one amenable* to biopsy (cohort A) 
OR 
ROLJRPHWDVWDWLF ERQH GLVHDVH  WR   DW URXWLQH ERQH VFDQ ZLWK DW OHDVW RQH OHVLRQ
amenable* to biopsy (cohort B) 
*e.g. safely to biopsy and expectably providing sufficient tissue yield 
5 
World Health Organisation (WHO) performance status 0 to 2 with no deterioration over the 
previous 2 weeks and minimum life expectancy of 12 months 
6 
No prior local and/or systemic treatment for localised prostate cancer 
7 
Willing to donate cancer tissue samples for research purposes (bone metastasis and 
primary tumour) 
Arm 2 
 
No 
Inclusion Criteria 
Patients are eligible only if they meet all of the following criteria 
1 
Male patients aged 18 years and older 
cT2-T3 / cN0-N1 / cM0 
Any Group Grade (GG) 2-5: this includes Gleason scores 3+4, 4+3, 4+4, 4+5, 5+3, 5+4, 
5+5. 
Any PSA  
2 
Histologically proven adenocarcinoma of the prostate 
  
3 
Planned radical prostatectomy as primary treatment for localised prostate cancer 
4 
World Health Organisation (WHO) performance status 0 to 2 with no deterioration over the 
previous 2 weeks and minimum life expectancy of 12 months 
5 
No prior local and/or systemic treatment for localised prostate cancer 
6 
Willing to donate cancer tissue samples for research purposes (any metastasis and 
primary tumour) 
 
 
 
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9.5 Exclusion Criteria 
Arm 1 
 
No 
Exclusion Criteria 
Patients will not be entered in the study for any of the following reasons 
1 
Involvement in the planning and/or conduct of the study (applies to staff at the study site) 
2 
Previous enrolment in the HYPROGEN study 
3 
As judged by the investigator, any evidence of severe or uncontrolled systemic disease 
(e.g. uncompensated respiratory, cardiac, hepatic or renal disease) 
4 
Evidence of any other significant clinical disorder or laboratory finding that made it 
undesirable for the patient to participate in the study 
5 
Any investigational agents or study drugs from a previous clinical study within 30 days of 
the first tissue collection 
6 
Prior treatment of localized prostate cancer including radiotherapy and/or androgen-
deprivation therapy 
7 
Judgment by the investigator that the patient should not participate in the study if the 
patient is unlikely to comply with study procedures, restrictions and requirements 
8 
Contra-indications to MRI (incl. pacemakers etc.)  
9 
Bone metastases in difficult to reach areas or areas which might be at risk for pathological 
fracture post biopsy as judged by biopsying radiologist / chief investigator 
10 
Increased risk of bleeding as a result of biopsy 
11 
History of bleeding disorders or thrombocytopenia (platelets <100/nL) 
12 
Concomitant treatment with anticoagulant therapy, e.g. warfarin/low molecular weight 
heparin or Anti-Xa-inhibitors and other NOACs, if temporary cessation medically not 
justifiable 
13 
Current urinary tract infection (UTI) or prostatitis 
Arm 2 
 
No 
Exclusion Criteria 
Patients will not be entered in the study for any of the following reasons 
1 
Involvement in the planning and/or conduct of the study (applies to staff at the study site) 
2 
As judged by the investigator, any evidence of severe or uncontrolled systemic disease 
(e.g. uncompensated respiratory, cardiac, hepatic or renal disease) 
3 
Any investigational agents or study drugs from a previous clinical study within 30 days of 
the first tissue collection 
4 
Prior treatment of localized prostate cancer including radiotherapy and/or androgen-
deprivation therapy 
5 
Judgment by the investigator that the patient should not participate in the study if the 
patient is unlikely to comply with study procedures, restrictions and requirements 
6 
Contra-indications to MRI (incl. pacemakers etc.)  
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9.6 Study Payment 
Patients will not receive any allowance for study participation including the provision of 
biopsy tissue or blood samples. However, a reimbursement of up to £40 per research 
visit may be requested to cover costs incurred for travel to research appointments for 
those patients within Arm 1 only. 
 
9.7 Informed Consent 
A member of the MDT will identify the patient at the MDT and approach the patient for 
possible inclusion into the study and provide the participant information leaflet either 
during a routine appointment to discuss the MDT recommendations or by post. All 
responsible consultant urological surgeons and clinical oncologists are subinvestigators 
of this study. Informed consent will be obtained appropriate members of the Christie 
urological research team at The Christie NHS Foundation Trust.   
All potential study participants will be given a participant information leaflet prior to 
informed consent. It should be made clear to the potential trial participant that consent is 
voluntary and can be withdrawn without giving reasons and without prejudicing their 
ongoing/future care.  
Potential study participants will be given adequate time to consider participation in the 
study based on the participant information leaflet.  If the patient is willing to participate, 
an appointment will be made at the Christie NHS Foundation Trust within 5 working 
days, where the informed consent form will be signed.  The patients¶ pimonidazole 
hydrochloride tablets will be dispensed from the Christie Pharmacy, if they have agreed 
to it and it is available. A research blood sample will also be taken at this visit. 
Consent will be taken by a researcher who is a GCP trained doctor and who has been 
delegated by the PI to undertake this activity (and this is clearly documented on the 
delegation log). No study-related activities can be undertaken prior to consent. 
The original, signed copy of the patient information sheet and consent form(s) will be 
retained in the Investigator Site File, with a copy in the patient notes. A copy will also be 
given to the participant.  
 
9.8 Ineligible and Non-recruited Patients 
Patients who consent to participate in the study, but who are later deemed ineligible 
during the screening process will be logged on the screen-failure log. Lack of inclusion in 
this study will in no way affect their ongoing/future clinical care. 
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9.9 Study Registration 
Patients who have consented to participate will be assigned a unique ID code by the 
HYPROGEN study co-ordinator. This ID code will be used throughout their participation 
in the study. The master list will be held at the Urology Research TeamV¶ office at The 
Christie NHS Foundation Trust. 
 
9.10 Withdrawal of Participants 
If at any time a patient expresses a wish to withdraw consent for ongoing study 
participation the following procedures will apply:  
x 
Withdrawal of consent will be clearly documented in the study documentation 
DQGWKHVWXG\SDUWLFLSDQW¶VPHGLFDOUHFRUG 
x 
No further clinical data will be collected from the study participant. However, 
existing clinical data held will be retained and used in the study. 
x 
Samples of tissue and/or blood already collected will be retained for use in the 
study. 
x 
Sample left-overs after all study-related assessments have been finished will be 
donated and stored anonymised at the Manchester Cancer Research Centre 
biobank. 
x 
7KHVWXG\SDUWLFLSDQW¶VSULYDF\ZLOOEHUHVSHFWHGDQGSUHVHUYHG 
The investigator may also advise that a patient discontinues study participation before 
undergoing the biopsies, should any untoward event occur. Reasons for investigator 
withdrawal include (but are not limited to): 
x 
Adverse events or acute onset of co-morbidities compromising the patient in 
having biopsies within the specified study criteria. 
x 
Any start of prostate cancer treatment prior to the first biopsy / blood samples 
being taken. 
x 
Severe non-compliance to this protocol.  
 
10 DATA MANAGEMENT 
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10.1 Case Report Forms 
Data will be collected on paper case report forms (CRFs) and then entered into an 
electronic database. The database will be located at The Christie NHS Foundation Trust, 
and only accessible from The Christie Intranet. The database will be password protected 
and accessible only to those delegated to input or review the data. 
 
10.2 Data Flow 
Each participant will be assigned a trial identity code number, allocated for use on CRFs, 
other trial documents and any electronic spreadsheet. These documents and database 
will use date of birth (dd/mm/yy) and patient number. 
CRFs will be treated as confidential documents and held securely in accordance with 
regulations. The investigator will make a VHSDUDWHFRQILGHQWLDOUHFRUGRIWKHSDUWLFLSDQW¶V
name, date of birth, local hospital number or NHS number, and Participant Trial Number 
(the Trial Recruitment Log), to permit identification of all participants enrolled in the trial 
in accordance with regulatory requirements and for follow-up as required. 
Completion of CRFs shall be restricted to those personnel approved by the Chief or local 
3ULQFLSDO,QYHVWLJDWRUDQGUHFRUGHGRQWKHµ7ULDO'HOHJDWLRQ/RJ¶ 
All paper forms shall be filled in using black or blue ballpoint pen. Errors shall be crossed 
out (but not obliterated by using correction fluid) and the correction inserted, initialled 
and dated. Corrections should be made legibly and initialled and dated by approved 
personnel. The reasons for significant changes must be provided. 
If any data are not available, omissions should be indicated on the case report forms. 
The NHS Code of Confidentiality will be followed for this study. 
Sensitive data stored on NHS or University computers will be encrypted to applicable 
NHS standards (AES256). The results of the study may be published in the medical 
literature, but patient identity will not be revealed. 
 
10.3 Storage of Imaging and Sequencing Data 
The data collected as part of the scanning procedures will be stored for fifteen years. 
The electronic data will be archived on computer systems managed by the Christie NHS 
Foundation Trust and the University of Manchester, and paper records will be stored in 
appropriately secure conditions. Although some personal (identifiable) data will be 
retained for up to five years from the day of the scan, most of the scan data itself will be 
anonymized shortly after the scan has taken place. 
Data from nucleic acid sequencing will be anonymized, encrypted and securely stored 
by SciCom and for a period of 10 years. 
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11 ADMINISTRATIVE AND REGULATORY DETAILS 
11.1 Study Sponsor 
The Christie NHS Foundation Trust will act as the sponsor for this study. Delegated 
responsibilities will be assigned to the Chief Investigator and HYPROGEN research 
team to manage the study on behalf of the sponsor.  
 
11.2 Funding Body 
Professor Robert G Bristow, PhD (Director of the Manchester Cancer Research Centre, 
MCRC) using funds from the FASTMAN Centre of Excellence award (Prostate Cancer 
UK) and CRUK MI Leadership funds. 
This study is an academic lead study that has been costed in accordance with DOH 
guidelines: Attributing the costs of health & social care Research & Development 
(AcoRD).  
The study has been adopted on to the NIHR portfolio providing access to service 
support costs.  
 
11.3 Peer Review 
The study design, protocol and methodology were independently reviewed, discussed 
and approved by Professor Robert Jones (Clinical Trials Unit Glasgow, Beatson West of 
Scotland Cancer Centre, Glasgow, UK) on behalf of the National Cancer Research 
Institute (NCRI) Prostate Clinical Study Group (CSG) and Dr Simon Chowdhury (Medical 
2QFRORJ\'HSDUWPHQW*X\¶VDQG6W7KRPDV¶1+6)RXQGDWLRQ7UXVW/RQGRQ8.. 
 
11.4 Patient and Public Involvement 
To provide easily understandable and patient centred study materials, e.g. participant 
information leaflet and consent form, a prostate cancer patient representative (RM, 
Stockton-on-Tees, UK) has reviewed and approved these documents and was involved 
in study design discussion. The patient is completely independent from the sponsor, 
funder and all investigators of the study. 
 
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11.5 Ethical Approval 
No patients will be entered onto the study before ethical approval has been confirmed. 
The study protocol has received the favourable opinion of the HRA Ethics Committee for 
the study protocol, informed consent forms and other relevant documents.  
The study will be conducted in accordance with, but not limited to, the Human Rights Act 
1998, the Data Protection Act 2018, Freedom of Information Act 2000 subject to the 
provisions of sections 41 and 43 thereof, the EU Clinical Trials Directive, the Human 
Tissue Act 2004, ICH GCP E6 R2 addendum, the Declaration of Helsinki 1996, Ionising 
Radiation Medical Exposure Regulations (IRMER), and the UK Policy framework for 
Health and Social Care research as amended from time to time. 
Where patients agree to take part in the study, they will be informed of how data are 
recorded, collected, stored and processed and may be transferred to other countries, in 
accordance with The General Data Protection Regulation (GDPR) (EU) 2016/679. As 
the sponsor of the study trial is a non-commercial organisation the legal basis for the 
KDQGOLQJDQGSURFHVVLQJRIGDWDLVµWDVNLQWKHSXEOLFLQWHUHVW¶ 
Annual progress reports will be submitted to REC within 30 days of the anniversary date 
on which the favourable opinion was given, and annually until the study is declared 
ended. 
 
11.6 Confidentiality 
All Investigators and site staff involved with the study must comply with the requirements 
of General Data Protection Regulations 2018 with regard to the collection, storage, 
SURFHVVLQJ DQG GLVFORVXUH RI SHUVRQDO LQIRUPDWLRQ DQG ZLOO XSKROG WKH $FW¶V FRUH
principles.  
Mr Wes Dale, Managing Director (Research, Innovation and Education) at The Christie 
NHS Foundation Trust will act as the custodian of the data collected in this study. Prof 
Rob Bristow will act as the custodian for the samples collected during the study. 
 
Key principles include: 
x 
The research team will have access to the clinical data for the purpose of this 
study. Study data may be inspected by individuals from the sponsor or 
regulatory authorities for monitoring and audit purposes. Informed consent will 
be obtained to cover this activity.  
x 
Any personal data recorded will be regarded as confidential, and any 
information which would allow individual patients to be identified will not be 
released into the public domain.  
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x 
Appropriate storage, restrLFWHGDFFHVVDQGGLVSRVDODUUDQJHPHQWVIRUSDWLHQW¶V
SHUVRQDO DQG FOLQLFDO GHWDLOV 3DWLHQW¶V QRWHV DQG VWXG\ ILOHV ZLOO EH KHOG LQ D
secure storage area with limited access. Computers used to collate data will 
have limited access measures via usernames and passwords.  
 
11.7 Protocol Compliance 
The study protocol must be adhered to at all times. Intentional deviations from the 
protocol are not permitted under any circumstances.  
Accidental protocol deviations must be reported to the Chief Investigator and the 
sponsor. Recurring deviations will be investigated by the sponsor to deem if it can be 
classed as a serious breach. 
Identified/suspected serious breaches of the study protocol or GCP will be notified to the 
Operational Director of Research, or Quality Manager via: 0161 918 7572, 
breaches@christie.nhs.uk.  
The R&D Sponsor Team will be notified of other deviations significantly impacting the 
risk profile of the study or significantly interrupting study services or supplies via: 0161 
918 7357,  christiesponsoredresearch@christie.nhs.uk.  
Minor deviations should be monitored by the research team for seriousness and trends. 
Sponsor shall maintain oversight of these through periodic sponsor oversight activity 
reports (SOAR) and review of trial management group minutes. 
 
11.8 Trial Monitoring 
A trial monitoring plan will be developed and agreed by the research team and the 
sponsor. The procedures and anticipated frequency will be based on the trial risk 
assessment.  
 
11.9 Study Oversight 
The safety of this study is being over seen by the Safety Review Committee (SRC) 
which will be under the chairmanship of the Chief Investigator and will comprise of the 
co-investigators and study co-ordinator. The committee will meet to review the study 
conduct over the first five and ten patients enrolled.  
A Trial Management Group (TMG) will be established and will include those individuals 
responsible for the day to day management of the study including the Chief Investigator, 
Co-investigators and study managers as detailed in the key study contacts section of 
this protocol. Meetings will take place after inclusion of the fifth and tenth patient and 
every 3 months thereafter in order to evaluate recruitment and feasibility of the study 
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interventions and to optimise study procedures by developing protocol amendments, if 
needed. 
Key findings/decisions from these meeting will be notified to the sponsor as soon as 
possible. 
Sponsor shall maintain oversight of the study conduct through  
x 
Reviews of Annual Progress Reports (APR).  
x 
Periodic sponsor oversight activity reports (SOAR) by the research team.  
x 
Reviews of Trial Management Group and Safety Review Committee minutes. 
 
 
 
 
11.10 Protocol Amendments 
Any changes in research activity will require an amendment and will be initiated by the 
Chief Investigator. Proposed changes must be submitted in writing to the sponsor. The 
amendment will be categorised as substantial or non-substantial.  
Any required changes to the documents that supported the original ethical approval will 
be submitted as an amendment to the appropriate ethical and regulatory authorities by 
the research team. Substantial amendments will not be implemented until the HRA and 
REC grant approval. 
)RUDQ\DPHQGPHQWVWKDWZLOODIIHFWDSDUWLFLSDWLQJVLWH¶VORFDOFDSDELOLW\DQGFDSDFLW\
the research team will confirm with each participating sites R&D department that local 
capability and capacity is ongoing.  
An Urgent Safety Measure (USM) may be put in place with immediate effect without 
gaining prior authorisation by the REC in order to protect clinical trial participants from 
any immediate hazard to their health and safety. USMs must be approved by the 
sponsor prior to implementation however REC and HRA must be notified by substantial 
amendment within 3 days of the measures being taken. 
 
11.11 Liability and Indemnity 
As the Chief Investigator (Prof RG Bristow) is a member of staff of the University of 
Manchester, study insurance will be covered under the University¶V SROLF\ IRU0HGLFDO
Malpractice Errors and Omissions. 
As the sponsor, The Christie NHS Foundation Trust, is an NHS organization, the NHS 
indemnity scheme will apply in the event of harm to participants arising from the conduct 
or management of the study.  
 
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11.12 Adverse Events and Adverse Reactions 
The study does not involve any drug/device treatment (androgen deprivation and/or 
chemotherapy is given after the end of study as part of clinical routine care or a 
subsequent clinical trial unrelated to this study) and all procedures performed are part of 
standard clinical practice. Pimonidazole hydrochloride is used as a hypoxia marker 
substance without any known side effects at the prescribed dose of 0.5 g/m2. 
Adverse events following the biopsy visit should be monitored as per standard of care by 
the local urology on call team at the study site where the biopsies have been taken.  
Adverse events do not need to be recorded on the CRF unless they affect study biopsy 
collection or study participation. Any adverse events which do not affect a patientV¶ 
participation in this study, will not be recorded.  
A Serious Adverse Event (SAE) is any untoward medical occurrence in a study patient 
that:  
x 
Requires inpatient hospitalisation or prolongation of existing hospitalisation.  
x 
Results in persistent or significant disability/incapacity, or  
x 
Is a congenital anomaly/birth defect  
x 
Is life threatening  
x 
Results in death 
We do not anticipate any serious adverse events. However, any AEs meeting the 
definition of a Serious Adverse Event (SAE) must be reported to the CI/delegated 
research staff using the trial specific SAE Report Form immediately, and within 24 hours 
of observing or learning about the event. As many sections of the SAE form should be 
completed as possible (with further information being provided as it becomes available). 
The CI will be responsible for reviewing the reports for: 
x 
Completeness  
x 
Causal link to the study procedures 
x 
Expectedness 
The 
Sponsor 
will 
be 
notified 
by the CI/delegated 
research 
staff 
through 
sae@christie.nhs.uk if an SAE is identified as related to the study and is unexpected 
(SUSAR). Identified SUSARs will be reported (by the CI/ delegated research staff) to the 
relevant REC within 15 days of becoming aware of the event, using the HRA Non-
CTIMP Safety Report Form. 
SAEs will only be collected up to 30 days after the last biopsy sample is taken. All SAEs 
must be followed-up until resolution and site investigators must provide follow-up SAE 
reports if the SAE had not resolved at the time the initial report was submitted.  
 
11.13 End-of-Study Notification 
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HYPROGEN - Hypoxia-driven metastatic prostate cancer genomics 
Page 40 of 41 
Protocol v4.0 08.04.2022 
IRAS no.: 262789            Sponsor ref. no.: CFTSp155            Christie ref. no.: 18_DOG04_201 
The end of study notification will be submitted in writing to the REC within 90 days of the 
date of the patient inclusion. In the event of early study termination, the notification will 
be submitted to the REC within 15 days. 
 
11.14 Study Summary Report 
The study team is responsible for compiling and submitting the final study report to the 
sponsor and REC within one year of study closure.  
 
11.15 Archiving 
Essential documents are documents that individually and collectively permit evaluation 
of the conduct of the trial and substantiate the quality of the data collected. Essential 
documents will be maintained at The Christie NHS Foundation Trust in a way that will 
facilitate the management of the study including audit. Essential documents, including 
personal data will be stored for up to 5 years after the end of the study. 
At the point where it is decided that the trial documentation is no longer required, the 
sponsor will authorise the destruction of all essential documents related to the 
HYPROGEN study. 
 
11.16 Publication Policy 
The data arising from the study will belong to the trial sponsor, The Christie NHS 
Foundation Trust.  
The main study results will be published in a peer reviewed journal, on behalf of all co-
investigators and collaborators. All presentations and publications relating to the trial 
must be authorised by sponsor. 
Participants will be given the option to request a copy of the study results should they so 
wish. 
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HYPROGEN - Hypoxia-driven metastatic prostate cancer genomics 
Page 41 of 41 
Protocol v4.0 08.04.2022 
IRAS no.: 262789            Sponsor ref. no.: CFTSp155            Christie ref. no.: 18_DOG04_201 
12 REFERENCES 
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Rankin, E.B. and A.J. Giaccia, Hypoxic control of metastasis. Science, 2016. 352(6282): 
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Rankin, E.B., J.M. Nam, and A.J. Giaccia, Hypoxia: Signaling the Metastatic Cascade. 
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Yang, L., et al., Development and Validation of a 28-gene Hypoxia-related Prognostic 
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Parry, M.A., et al., Genomic Evaluation of Multiparametric Magnetic Resonance Imaging-
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Danila, D.C., et al., Circulating tumor cell number and prognosis in progressive 
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Kuske, A., et al., Improved detection of circulating tumor cells in non-metastatic high-risk 
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Ulz, P., et al., Whole-genome plasma sequencing reveals focal amplifications as a driving 
force in metastatic prostate cancer. Nat Commun, 2016. 7: p. 12008. 
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Wyatt, A.W., et al., Concordance of Circulating Tumor DNA and Matched Metastatic 
Tissue Biopsy in Prostate Cancer. J Natl Cancer Inst, 2017. 109(12). 
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Romanel, A., et al., Plasma AR and abiraterone-resistant prostate cancer. Sci Transl 
Med, 2015. 7(312): p. 312re10. 
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Wyatt, A.W., et al., Genomic Alterations in Cell-Free DNA and Enzalutamide Resistance 
in Castration-Resistant Prostate Cancer. JAMA Oncol, 2016. 2(12): p. 1598-1606. 
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Azad, A.A., et al., Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: 
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de Bono, J.S., et al., Randomized Phase II Study of Akt Blockade With or Without 
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Pritchard, C.C., et al., Inherited DNA-Repair Gene Mutations in Men with Metastatic 
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