Circulating Tumour Cells as Biomarkers to Predict Prostate Cancer Metastasis for Treatment Stratification of Localised Cancer
Conditions: Prostate Cancer
Sponsor: Queen Mary University of London

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
Full Title  
Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment 
stratification of localised cancer 
 
  
 
Short Title 
C-ProMeta-1 
 
Sponsor 
Queen Mary, University of London  
 
Contact person: 
 
Dr Mays Jawad 
Research & Development Governance Operations 
Manager 
Joint Research Management Office 
 
5 Walden Street 
 
London 
 
E1 2EF 
 
Phone: 020 7882 7275/6574 
 
Email: sponsorsrep@bartshealth.nhs.uk 
 
IRAS Number 
140998 
 
Sponsor (ReDA) Number 
N/A 
 
REC Reference 
 
 
19/LO/0994 
HTA License organization           Barts and the London Queen Mary’s School of 
Medicine and Dentistry 
Designated Individual                  Ms Katie Ersapah 
License number                          12199 
Sub-protocol investigators 
Prof Yong-Jie Lu & Mr Greg Shaw &  
Prof Rhian Gabe 
 
Professor Yong-Jie Lu  
Professor in Molecular Oncology  
Barts Cancer Institute  
Queen Mary University of London 
Email: y.j.lu@qmul.ac.uk 
T: 020 7882 3597 
 
 
  
Mr Greg Shaw  
Consultant Urologist 
   Institute of Urology 
      University College London Hospitals NHS Trust 
      Email: gregshaw@nhs.net 
 
Prof. Rhian Gabe 
Professor of Clinical Trials and Biostatistics 
   Centre for Cancer Prevention 
   Wolfson Institute of Preventive Medicine  

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
   Queen Mary University of London  
   T: 02034567890 
 
 
List of Collaborators 
 
 
Professor Daniel Berney, Consultant Pathologist 
and Academic lead of the Robert Lane Orchid 
Tissue Bank (RLTB), Centre for Biomarker and 
Biotherapeutics, Barts Cancer Institute, Queen 
Mary University of London 
 
Dr. Prabhakar Rajan, Consultant Urologist, Barts 
Cancer Institute, Queen Mary University of London 
 
Dr. Justin Collins, Consultant Urologist, Institute of 
Urology, University College London 
 
 
List of laboratories 
Centre for Biomarkers & Biotherapeutics  
 
Barts Cancer Institute 
First floor, South Wing 
John Vane Science centre  
Charterhouse Square 
London 
EC1M 6BQ 
 
 
Contents 
 
1. Glossary/Abbreviations: 
ADT 
Androgen Deprivation Therapy 
AUC  
Area under the ROC Curve 
BCI 
Barts Cancer Institute 
BCR 
Biochemical Recurrence 
CTC 
Circulating Tumour Cell 
CRF 
Clinical Research Fellow 
EAU 
European Association of Urology 
EDTA  
Ethylenediamine Tetraacetic Acid  
FTE 
Full Time Equivalent 
GCP 
Good Clinical Practice 
ID 
Identification 
Min 
Minute 
MHRA 
Medicines and Health-care Products Regulatory 
Agency 
MTA 
Material Transfer Agreement 
PBS 
Phosphate Buffered Saline 
PBMC 
Peripheral Blood Mononuclear Cell 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
PCa 
Prostate Cancer 
PI 
Principal Investigator 
PIS 
Patient Information Sheet 
PSMA-PET 
Prostate Specific Membrane Antigen – Positron 
Emission Tomography  
QMUL 
Queen Mary University of London 
RBC 
Red Blood Cells 
RT 
Room Temperature 
ROC 
Receiver Operating Curve 
RP 
Radical Prostatectomy 
RLTB 
Robert Lane Tissue Bank 
SOP 
Standard Operating Procedure 
TBAO 
Tissue Bank Acquisition Officer  
UCLH 
University College London Hospital 
WBC 
White Blood Cells 
 
2. Introduction:  
2.1. 
Rational: As many prostate cancer (PCa) cases diagnosed at early stage are 
indolent, PCa progression risk- stratification systems have been developed, based 
on biopsy characteristics, staging and PSA levels, and patients are classified into 
low- intermediate- and high-risk groups for treatment stratification. However, these 
systems are imperfect. Moreover, determining which curative treatment will be 
most effective for individual patients with localised PCa is challenging and remains 
a major obstacle in improving patient management and is recognised as an unmet 
clinical need. Cancer metastasis is the main cause of PCa death and determines 
the selection of therapeutic methods. Theoretically, localised PCa should be cured 
by radical prostatectomy (RP). However, more than half of apparently localised 
high-risk PCa cases treated by RP reoccur, indicating the presence of undetectable 
micro-metastasis at the time of surgery. These particular patients require additional 
treatment, 
usually 
with 
radiotherapy 
and 
systemic 
hormone 
therapy.  
The major challenge in managing aggressive localised PCa is differentiating truly 
localised PCa from those with micro-metastatic disease, which cannot be cured by 
RP. Pelvic lymph node dissection for high-risk localised disease is the current 
diagnostic test for assessing spread of disease but is invasive and performed 
during surgery. A test is required which can be performed before surgery to 
distinguish patients suitable for RP from those who would benefit from more 
extensive systemic treatment and radiotherapy. No current imaging test has the 
resolution to detect micro-metastases that may consist of just a few viable PCa 
cells. Circulating tumour cells (CTCs), the seeds of cancer metastasis, can occur 
at a very early stage of cancer development. We hypothesise that detection of 
CTCs provides an accurate indicator of cancer micro-metastasis and CTC gene 
expression may predict the potential of future metastasis development. We have 
established a promising CTC analysis method, by which we have detected CTCs 
in all patients with metastatic PCa and for the first time have demonstrated the 
value of using CTC analysis in predicting clinically significant post-biopsy clinically 
significant PCa outcome in pre-biopsy patients. Our CTC results may reflect the 
existence of micro-metastasis and predict PCa progression risk better than the 
current imperfect risk-stratification systems. Therefore, a prospective study with 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
clinical outcome follow- up over a long period is required to confirm the value of 
this CTC analysis in determining micro- metastasis, i.e. predicting post-RP PSA 
failure and future metastasis development. 
 
2.2. 
Aims: 
 
I. 
To establish the value of CTC positivity in predicting post-RP treatment failure, 
including BCR and new lesions detected by cancer imaging. 
 
II. 
To establish the value of a combined CTC test (including CTC enumeration and 
gene expression) in predicting post-RP treatment failure and the time of the 
development of BCR and new lesions detected by cancer imaging.  
 
III. 
To provide a preliminary assessment of the prognostic value of CTC data (positivity 
and gene expression) in predicting post-RP PCa metastasis development, overall 
survival and cancer specific survival and the generation of CTC data for future 
longer-term follow-up.  
 
IV. 
To provide evidence and data which can be used to design a future clinical trial of 
a CTC-based intervention to be used to guide therapeutic choice in the 
management of high-risk localised PCa. 
 
3. Summary and synopsis 
This study will require 10-year patient follow up for analyses of the primary and 
secondary endpoints to have statistically significant power. Nevertheless, extensive 
research activities will be mainly conducted in the first 5-years where research funding 
has been secured. After the first 5-years, further research activity will only include 
annual patient clinical follow-up, data collection and statistical analysis. A renewal 
application for further funding will be submitted by the end of the first 5-year funding 
period. The overall study set up is summarised in Table 1. 
 
Table 1. Overall study summary. 
 
Methodology 
Single site, double-blinded, prospective, paired cohort study. Participating 
patients and clinicians involved in treatment or management will be blinded to 
the CTC results, to avoid influencing standard patient treatment, management, 
and progression outcomes after RP. 
Study objectives 
and endpoints 
 
Objectives are set in the context of high-risk patients diagnosed with localised 
prostate cancer scheduled for robot-assisted RP who have not had neoadjuvant 
ADT, and are listed below. 
Primary objectives: 
To establish the value of CTC positivity in predicting post-RP treatment failure, 
including BCR and new lesions detected by cancer imaging.  
To establish the value of a combined CTC test (including CTC enumeration and 
gene expression) in predicting post-RP treatment failure and the time of the 
development of BCR and new lesions detected by cancer imaging.  
Secondary objectives 
To investigate the potential of CTCs in predicting positive lymph node 
metastasis, which affects the selection and scale of RP as the initial treatment. 
To assess potential for convenient non-invasive disease monitoring 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
To provide a preliminary assessment of the prognostic value of CTC data 
(positivity and gene expression) in predicting post-RP PCa metastasis 
development, overall survival and cancer specific survival. 
To provide evidence and data which can be used to design a future clinical trial 
of a CTC- based intervention to be used to guide therapeutic choice in the 
management of high-risk localised PCa. 
To evaluate the longer-term prognostic value of CTC detection and CTC gene 
expression for metastases and cancer-specific survival.  
 
Primary endpoint 
The primary outcome is post-RP treatment failure during the first 4.5 years of 
follow up from start of recruitment. 
 
Post-RP treatment failure is defined as a PSA ≥ 0.2mg/ml at the routine PSA test 
3 months after RP (commonly called ‘failure to nadir’) and remaining at this level 
or further increase afterwards without further treatment, or imaging detected 
appearance of cancer lesions.  
 
Cancer lesions detected by imaging without a PSA rise might include 
neuroendocrine PCa and lesions detected by PSAM-PET. This combined post-
RP treatment failure primary endpoint will maximally capture all the clinically 
significant cancer appearance events. 
 
Secondary endpoints 
BCR during the first 4.5 years of follow up: PSA ≥ 0.2ng/ml at any time post-RP 
and remaining at this level or further increase afterwards without further 
treatment. 
Metastasis (any location)-free survival during the first 4.5 years of follow up. Only 
5% of subjects with distant metastasis event (based on traditional imaging 
technologies) within this time frame (4-6). 
Metastasis (any location)-free survival at 10 years follow up. to confirm that 
metastatic event rates have increased among the positives, i.e. a declining rate 
of "false positives". 
Deaths from any cause during the first 4.5 years of follow up 
Overall survival at 10 years of follow up. 
Prostate cancer specific deaths during the first 4.5 years of follow up. Expected 
to be 2% or less based on previous studies in the post RP context. 
Prostate cancer specific survival at 10 years of follow up. Our hypothesis is that 
most if not all of the patients dying of PCa will be in the CTC positive group. 
Target sample size 
294 
Eligibility 
Inclusion criteria: 
High/High intermediate risk non-metastatic risk localised PCa based on the 
EAU stratification system 
Scheduled for robot-assisted RP  
Informed consent  
 
Exclusion criteria:  
With other cooccurring cancers  
Neo-adjuvant ADT 
Adjuvant ADT  
Statistical 
methodology 
and 
analysis 
(if 
applicable) 
The primary analysis is the estimation of the sensitivity of the pre-surgery CTCs 
for detection of RP treatment failure, i.e. BCR or progression detected by 
imaging. Observed specificity, positive and negative prediction values will be 
reported. Prevalence of events and specificity will be presented over time. BCR-
free survival will be estimated and Kaplan-Meier curves presented. Preliminary 
investigation of the prognostic value of pre-surgery CTC data in terms of 
predicting survival outcomes will be investigated. CTC data will be explored 
alongside other pre-surgery risk factors using multivariate analysis. Recurrence 
events and follow up CTC results will be reported and compared over time.  

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
Study duration 
The planned maximum duration is 12 years 
(Target duration for patient recruitment is 2 years. Each patient provides blood 
samples for CTC analysis and target duration for completion of CTC analysis is 
1 year after end of recruitment. Target duration of long-term patient follow-up 
from point of recruitment is 10 years) 
        
4. Study design (first five years) 
The study flow diagram for the first 5 years is shown in figure 1. 
 
Figure 1. The study flow diagram for the first 5 years. 
 
The 5-year funded study will be taken with the planned milestones described in Table 2 
below. 
Table 2: Planned study milestones: 
Patient recruitment 
years 0-2 
Blood sample collection 
years 0-2.25 
CTC analysis 
years 0-3 
Follow up 
years 0-4.5 
Analysis 
years 4-5* 
*The statistical analysis will start after 4.5 year and takes 6 months to complete within the initial 5-
year funded period. 
 
5. Study population 
Potential participants will be identified, approached and screened by the study team if they 
are interested in participating as described in the study design above. In order to recruit 
patients meeting the eligibility criteria, it will be necessary to screen all patients in the 
participating centre who are diagnosed with high risk localised PCa based on the current 
European Urology Association classification system who have been deemed eligible for 
radical prostatectomy by a specialist multi-disciplinary committee, and who have been 
scheduled for surgery to completely remove the cancer in the prostate gland.  
 
Based on past patient numbers at UCLH, it is anticipated that approximately 600 patients will 
meet these initial criteria over the 2-year recruitment period. However, to meet our target 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
recruitment we anticipate contacting approximately 490 of these potential participants (Figure 
1). Potential participants will be identified via MDT meetings and in clinics. Potential 
participants who meet eligibility criteria will be posted, emailed or physically provided with a 
patient information sheet (PIS) approved by the ethics committee, prior to their next clinic 
appointment in order to give sufficient time to consider the information. At their clinical 
consultation (remote or face to face), they will be approached regarding the study, and given 
the opportunity to ask any questions and confirm eligibility checks. Inclusion and exclusion 
criteria are given below. 
 
Inclusion criteria 
 High/high intermediate risk non-metastatic PCa based on the EAU stratification 
system 
 Scheduled for robot-assisted RP 
 Informed consent 
 
Exclusion criteria 
 
With other cooccurring cancers  
 
Neo-adjuvant ADT 
 
Adjuvant ADT 
 
Withdrawal Criteria 
 
Unable or unwilling to undergo RP 
 
Patient changes their mind and withdraws their consent 
 
With an anticipated drop out of 40% due to either use of neo-adjuvant ADT or failure 
to consent in patients sent the PIS, we further anticipate 294 patients will contribute 
blood samples prior to RP. There is a target of 200 blood samples from eligible 
participants, who subsequent to having had RP do not have a positive surgery margin 
or other evidence of locally remaining disease, and do not subsequently have ADT 
(adjuvant ADT) or radiotherapy. It is intended that recruitment will continue until this 
target has been met. 
6. Patient recruitment, sample collection, lab CTC analysis and follow-up 
procedures 
Patients will be recruited (months 1-24) at UCLH, where the UK largest urological surgery 
centre is located and performs robot-assisted RP on PCa patients referred from several 
regional hospitals. The clinical team at UCLH will identify eligible patients who will be 
approached by the clinical research fellow (CRF) employed on the research project or the 
clinical care team for informed consent using the consent forms specifically designed for this 
project for blood collection and future research. Non-metastatic disease will be based on the 
current standard diagnostic imaging methods including CT/MRI and PSMA-PET/bone scan. A 
pre-surgery PSA test will be performed routinely at UCLH.  
 
2 x 10 ml blood samples will be collected (months 1-27) using the lavender cap EDTA tube 
according to our established method from each consented patient by the CRF or the clinical 
care team at UCLH during the pre- and post-RP PSA test blood sampling, and taken to the 
laboratory at Barts Cancer Institute, John Vane Science Centre, Charterhouse Square either 
by the CRF, a tissue bank acquisition officer (TBAO)(in the absence of the CRF) or the postdoc 
(anonymise samples transfer in the absence of CRF and TBAO) under the signed material 
transfer agreement (MTA), at room temperature. The samples will be transported in 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
designated sample carrier using a taxi service. No public transport is to be used for moving 
samples between sites.  
 
Each patient will be allocated a unique ID and will be used by the lab research team. This 
unique ID will be recorded in the tissue bank database for future correlation analysis of the 
CTC results with clinical follow-up data. Sample processing will be performed in tissue culture 
hood in the designated lab. The whole blood sample will be processed within 4hrs for CTC 
analysis after collection. Standard Operating Procedure (SOP) (C-ProMeta-1) that is 
generated 
describing 
sample 
process 
and 
analyses 
need 
to 
be 
followed.  
There will be fewer patients for the post-RP than the pre-surgery blood collection based on 
patient exclusion criteria after RP. 
 
Based on the sample size calculation 200 patients will be recruited for both pre- and post-
surgery blood sample collections for CTC analysis, which are expected to be achieved within 
the first two years of the project.  
 
Patient informed consent (including the permission to conduct remote longer-term follow-up) 
and blood sample collection will be performed by the CRF or the clinical care team.  
 
All patients will have a PSA test at 3 months after their RP during a routine follow-up at UCLH. 
A PSA ≥ 0.2 will be recorded as a treatment failure event and PSMA-PET scans will be 
performed as standard clinical care at the study hospitals to detect small PCa lesions. After 3 
months, patients are referred back to their original referring centres. Patients with negative 
PSA results at three months will be followed up by telephone or online app by the study CRF 
in order to ascertain whether subsequent PSA tests are positive (PSA ≥ 0.2), indicating BCR, 
metastasis occurs as detected by cancer imaging including PSMA-PET scan or if they have 
received salvage treatment with hormones/radiotherapy. In addition, details of long term follow 
up, such as post-operative PSA results and record of adjuvant therapies, will sourced from 
patients’ hospital electronic medical records, or requested from their local GP or health centre. 
all patients will be followed-up until the end of the project for metastasis progression as 
detected by imaging, PCa-specific death and death from any causes. In addition to data 
collected within the 5-year funded period, we plan conduct remote longer-term follow up with 
fully informed consent from patients at point of enrolment, including:  
 Contacting patients on an annual basis for 10 years so they may report their PSA 
and relevant further treatments. Methods of contact will be decided upon with PPI and 
may consist of list of preferred options including phone call by the CRF and/or use of 
secure electronic means via automated email prompts, secure website or app data 
entry. Mr. Shaw is currently collaborating with primary care colleagues to develop a 
patient support app under a separate funding support.  
 Flagging and linkage to the patients’ relevant electronic health data using their NHS 
number and date of birth. Under the Confidentiality Advisory Group (CAG), we will 
request treatment, imaging and cancer outcome data from NHS Digital based on ONS-
HES-DID (Data Imaging Data) and NCRAS data sets.  
 
We would thus be able to plan analyses of the diagnostic accuracy and predictive value of the 
CTC-based tests in terms of metastases and progression-based outcomes at 7.5 years and 
10 years of follow-up. Further longer-term follow-up after the 10-year will be performed by 
accessing the GP and NHS digital records if required and with further ethical approval. 
Patient recruitment, sample collection and follow-up procedure were summarised in Table 2 
Table 2. Patient recruitment, sample collection and follow-up procedure 
Procedure 
Screenin
g/Baselin
e 
Pre-
RP 
3 months 
post op 
12 month 
post op 
24 month 
post op 
… 
each 
year… 
120 month 
post op 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
Eligibility 
assessment 
x 
 
 
 
 
 
 
Informed 
Consent 
x 
 
 
 
 
 
 
Blood 
Sample 
collection  
 
x 
x 
 
 
 
 
PSA 
 
x 
x 
x 
x 
x 
x 
Medical 
record review 
and 
online 
app/phone 
follow-up 
(PSA and/or 
Adjuvant 
therapies) 
rates of BCR, 
metastasis, 
other 
progression 
events 
and 
death 
 
 
 
x 
x 
x 
x 
 
7. Evaluating the efficiency of CTC positive score and the gene panel in predicting 
RP treatment failure.  
Accuracy of the CTC scores alone and in combination with CTC gene panel expression data 
in determining RP treatment failure, i.e. BCR and/or metastasis via imaging, in particular 
PSMA-PET, over the study period will be measured. The prognostic value of CTC data will 
also be investigated for BCR, PSMA-PET detected cancer lesions, metastasis event, overall 
survival and cancer specific survival outcomes (see below statistical analysis plan section). At 
the end of this project, we expect to generate robust data to determine whether CTCs 
detectable in patients with high-risk localised PCa at pre- and post-RP can predict RP 
treatment failure and a CTC gene panel will further enhance the prediction value of CTCs for 
post-RP cancer progression.  
 
Statistical methodology and analysis 
The primary analysis is the estimation of the sensitivity of the pre-surgery CTCs for 
detection of RP treatment failure, i.e. BCR or progression detected by imaging. 
Observed specificity, positive and negative prediction values will be reported. 
Prevalence of events and specificity will be presented over time. BCR-free survival will 
be estimated and Kaplan-Meier curves presented. Preliminary investigation of the 
prognostic value of pre-surgery CTC data in terms of predicting survival outcomes will 
be investigated. CTC data will be explored alongside other pre-surgery risk factors 
using multivariate analysis. Recurrence events and follow up CTC results will be 
reported and compared over time.  
 
Statistical considerations 
1). Definitions: 
 
CTC score and result: CTCs will be identified based on the immunofluorescence 
staining as CK+/VIM−/CD45−, CK+/VIM+/CD45−, and CK−/VIM+/CD45− CTCs as 
described above in lab procedure Part IV. Positive CTC score were defined based on 
our 
previous 
data 
as 
any 
CK+/VIM+/CD45−, 
CK+/VIM+/CD45− 
and/or 
>3 
CK−/VIM+/CD45− cells identified in the PCa patients.  
 
CTC gene-expression result: a binary (negative/positive) result based on the gene-
expression profile from within the blood sample indicating the amount of genes 
expressed in CTCs from the PCa patients. 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
 
Combined CTC result: a binary (negative/positive) result based on both CTC number 
and gene-expression profile where a positive result indicates a patient should be 
considered to have high metastatic potential. 
 
MRI score: Multi-parametric MRI Likert scale 1-5 where >3 indicates agressive cancer. 
 
BCR: PSA ≥ 0.2ng/ml at any time post-RP and remaining at this level or further 
increase afterwards without further treatment (see Table 4 for endpoint definitions) 
 
Post-RP treatment failure: (see Table 4 for endpoint definitions) 
 
2). Sample size  
The study hypothesis is that the presence of CTCs has close to 100% sensitivity and 
specificity for micro-metastasis, and thus RP treatment failure. Due to the long-term 
nature of recurrence events, there will be a proof of principle part to this study which 
occurs within the funded study timeframe of 5 years, and an intention to collect and 
analyse relevant longer-term events at 10 years (see section 4 for objectives and 
endpoints). The primary endpoint is post-RP treatment failure, the majority of which 
will be due to BCR. Despite the long-term nature of events of interest, it will be possible 
to provide a timely estimate of sensitivity, since of those who do have an observable 
BCR event, most if not all are expected to have a positive CTC result. Thus, consistent 
with previous work, this study estimates predicting post-RP cancer events with 95% 
sensitivity. During the first 4.5 years of follow up, the hypothesised prevalence of BCR 
events to be at least 40%.  
 
In order to confirm an expected sensitivity of 95% within 5% precision either side of a 
95% confidence interval (CI) in a sample with an underlying prevalence of 40%, 
requires at least 73 events, that is at least 183 recruited individuals. Inflate this by 8.5% 
to a target recruitment of 200 to ensure suitability of samples and measurable 
outcomes. 
The recruitment plan will be 200 patients in the first two years of the 5-year project to 
allow an approximate 3.5 to 4 year median follow up. Specificity is important 
considering the future circumstance of potentially denying patients curative surgery, if 
a CTC test indicates metastasis. A study size of 200 and 120 true negatives would 
give a 97.5% lower confidence limit on a specificity of 99% (1% false positive rate). 
However, it is anticipated the specificity will be low due to positive results for those who 
recur beyond the 5-year study. Assuming these events are due to micro-metastases 
at the time of surgery, observed sensitivity and specificity will be used, plus additional 
analyses assuming up to 20% of the events occur after the funded 5-year project4-11 to 
estimate the real false positive and negative rates. 
 
3). Method of analysis  
Study flow and patient characteristics will be reported. Measures of accuracy 
will be presented with 95% confidence intervals (CIs). The primary analysis is 
the estimation of the sensitivity of the pre-surgery existence of CTCs for 
detection of RP treatment failure, i.e. BCR or progression detected by any 
imaging at the 4.5-year follow-up time point. Observed specificity, positive and 
negative prediction values will also be reported at 4.5 years of follow up, with a 
note to indicate that these estimates are subject to change given the long-term 
nature of recurrence events. Specificity will be presented over time and 
additional analysis accounting for artificial inflation of false positives based on 
increasing prevalence of events. BCR-free survival will be estimated and 
Kaplan-Meier curves presented.  
 
The number of participants with recorded metastases, lymph node metastases, 
deaths from any cause and PCa deaths during the first 4.5 years of follow up 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
will be reported alongside the number of these participants with a positive by 
CTC result, and positive combined CTC result. Metastases-free, PCa-specific 
and overall survival will be reported during the 5 years study time frame and it 
is intended to perform a later analysis at 10 years of follow up. Preliminary 
investigation of the prognostic value of pre-surgery CTC data in terms of 
predicting survival outcomes will be investigated using Cox regression, with 
hazard ratios reported with associated 95% CIs. CTC data will be explored 
alongside other pre-surgery risk factors such as plasma PSA using multivariate 
analysis. Recurrence events and follow up CTC results will be reported and 
compared over time. The above analyses will be applied to the CTC gene 
panel. The gene panel will also be correlated with the pre- and post- RP CTC 
number changes and cancer progression events. 
 
CTC results will be presented for the whole cohort who provided analysable 
blood samples and stratified by those included in the above analyses, and those 
excluded due to reasons such as a positive surgery margin, other evidence of 
locally remaining disease, and who subsequently have adjuvant ADT or 
radiotherapy. 
 
8. Ethics 
The study is a spin out project of the parent project authorised under RLTB ethics, 
where the main research tissue bank approval provided by the London City & East 
Research Ethics Committee. 
 
9. Data handling, storage and record keeping- to describe who and how the data will 
be handled, which data nodes will be used, where it will be stored and security of the 
data 
The CRF (funded by QMUL, but with contract at UCLH) will be part of the study 
management group and will act as the contact point between UCLH and QMUL. Under 
the MTA and data sharing agreement (DSA) between the two institutions and with 
patient consent, the CRF will collect blood samples during pre-surgery operation and 
after the surgery during the first PSA follow-up visit at UCLH. 
 
The following clinical data will be collected for each patient by the CRF: 
 
Histology reports  
 
Clinical stage 
 
Prostate Specific Antigen (PAS) (pre- and post-surgery)  
 
Imaging (MRI, PSMA-PET/CT and bone scan) 
 
Results and record of adjuvant therapies  
 
Treatment date and method for post-surgery cancer recurrence 
 
The data will be obtained using either electronic medical records at UCLH NHS Trust 
system or will be requested from patient’s local GP or health centres. PCa recurrence, 
metastasis and the starting of new treatment will also be directly collected from the 
patients by phone or secure online system/app during the post-operation follow-up 
period by the CRF. Collection of follow up data from GP and NHS digital records will 
be with approval from the Confidentiality Advisory Group (CAG) 
 
Each patient will be assigned a unique ID, and this ID will be used for identifying the 
patient. All samples will be anonymised using this ID. Completed consent forms will be 
handed over to the RLTB for secure storage. Patient identifiable data and the above 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
clinical data will be held securely in a file designated for the study in the RLTB folder 
in the Barts Cancer Centre server. The file will be maintained and updated by the RLTB 
data administrator and the CRF. The file will be password protected and only be 
accessible to authorised staff, inclusive of RLTB manager, study PI, RLTB data 
administrator and the CRF.    
 
A sample receipt form will be completed by the research team upon receipt of sample 
to the Barts Cancer Institute. The time of sample receipt to research site, the condition 
it is received and where they are stored will be recorded separately which will be 
accessible to research team. Location of sample will be recorded in item tracker for 
traceability purposes as a separate arm under RLTB storage. 
 
The data generated from this study will include immunofluorescence images of CTCs, 
RNA expression data of the metastasis associated genes in CTCs and follow-up data 
of clinical and cancer progression associated to PCa patients. These data will also be 
securely stored in a designated folder in the BCC IT server with access to PI and his 
research team. The data will be curated throughout its life-cycle (during the study and 
before the data is published). Each Hyzip (a CTC multiplex gene expression analysis 
system at Angle) gene expression value will be recorded in the original data format 
and saved in the above designated folder in BCC server and will be available for data 
sharing after the publication.  
 
With relevant safeguards applied for intellectual property and publications have been 
done of the main findings, anonymised data of the findings will be publicly accessible.  
These research data of the main outcomes, will be included in an excel spread sheet 
located in the above-mentioned designated folder in the server. This file, with relevant 
ethical, legal and institutional policies and regulatory permissions, will be made 
available through a web link published in the scientific journals and/or the BCI Institute 
website.  
 
The electronic data will be securely stored in the BCC server for 25 years. If required, 
the Arkivum long-term data archival service, which is used by the institute will be used.  
The clinical information asset will be registered in our institute IT system using the Data 
Asset Register Toolkit (DART). Paper records will be archived using the QMUL archive 
system and stored for 25 years. 
 
Data obtained for each patient and generated through the research project will be 
handled in accordance to the Data Protection Act 2018 UK and in reference to the 
General Data Protection Regulation (GDPR). All staff that will be involved with the 
project that handle data will have relevant training prior to handling and managing the 
data. 
 
 
10. Safety procedures if there’s any adverse events or deviations from the process  
Obtaining blood samples for this study will include the normal associated risks with 
venepuncture, such as temporary discomfort and bruising. Blood samples for pre-
operative preparation and pre-and post-operative PSA monitoring is part of the NHS 
standard of care for PCa patients, so obtaining samples for this study will not involve 
any additional risk or burden for patients.  
The study findings will not affect patient care in any way. All treatment management 
decisions for patients will be made by the clinical team unrelated to the study. 
 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
11. Monitoring and auditing- to briefly describe how any changes will be monitored and 
recorded and if any auditing will be taking place 
 
The Sponsor, funding body and/or regulatory bodies may audit the study, study site or 
central facility.  
 
The PI will ensure there are adequate quality and number of monitoring activities 
conducted by the study team. This will include adherence to the protocol, procedures 
for consenting, and source data verification to ensure adequate data quality.  
The PI will inform the Sponsor should they have concerns which have arisen from 
monitoring activities, and/or if there are problems with any oversight or monitoring 
procedures. 
 
RLTB will be auditing consent forms on receipt and study auditing will be carried out 
as per local RLTB policy with prior agreement with the study team.  
 
12. Study management and oversight for the spin off project and also feedback to 
RLTB committee 
The study’s management group (SMG), consisting of the CI, PIs/Co-PIs, key 
collaborators, the CRF and statistician will convene on a monthly basis. The CRF and 
study manager will update the RLTB steering committee annually of the project 
progression and any feedback received will be communicated back to the SMG. 
 
The Study Steering Committee (SSC), which includes a patient member, a consultant 
urologist (chair), a biomarker study specialist/statistician and a basic research scientist 
independent from the study will be formed to monitor recruitment and study progress 
in terms of outcome data collection and statistical assumptions underpinning the study, 
including confidential review of the event rates. 
 
13. Finance-who is funding the spinoff study and what is funded 
The spin-off study is funded by Prostate Cancer UK (PCUK) initially for 5 years and 
will be renewed with separate application at end of the 1st 5-year period with review 
from the funding body. The first 5-year PCUK funding will include salary for the CRF 
(70% FTE to work on this project), postdoctoral research assistant, 55% salary for 
study manager, 20% salary for data administrator, 40% salary for a statistician (last 3 
of the 5 years) as well as consumables.  Parsortix CTC isolation systems and Hyzip 
multiplex CTC gene expression system, are covered by Angle. 
 
14. Any insurance and indemnity for participants 
The insurance that Queen Mary University of London has in place provides cover for 
the design and management of the study as well as "No Fault Compensation" for 
participants, which provides an indemnity to participants for negligent and non-
negligent harm. 
 
 
15. Dissemination of research findings 

Protocol for spin off research project- Circulating tumour cells as biomarkers to 
predict prostate cancer metastasis for treatment stratification of localised cancer 
Sub-protocol version 1 
 
14/07/2021 
To ensure the results and impact of our research are widely communicated to their 
intended beneficiaries, we will disseminate them via: 
a) High impact peer-reviewed publications with open access; 
b) Scientific conferences and seminars; 
c) Communication with the general public (fundraisers and schools); 
d) Press releases (our previous researches were successfully communicated in 
TV/newspapers/online media); 
e) Summaries of study progress and clinical relevance as feedback to participating 
patients; 
f) Interaction with the relevant healthcare authorities, such as MHRA, to translate the 
research findings into clinical use.