An Observational Study to Correlate the Results of Ploidy and Stroma Analysis With Prognosis in Early Rectal Cancer
Conditions: Rectal Cancer
Sponsor: Oxford University Hospitals NHS Trust

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
 Page 1 of 16 
 
Study Title:  An observational study to correlate the results of ploidy and stroma analysis with 
prognosis in early rectal cancer 
Internal Reference Number / Short title: Ploidy and stroma in early rectal cancer 
Ethics Ref: 16/WM/0443 
Date and Version No: v1.1 21 October 2016 
 
Chief Investigator: 
Chris Cunningham, Oxford University Hospitals NHS Foundation Trust 
Investigators:  
Helen Jones, Oxford University Hospitals NHS Foundation Trust 
David Kerr, Oxford University Hospitals NHS Foundation Trust 
Håvard Danielsen, Institute for Cancer Genetics and Informatics, Oslo 
Hanne Askautrud, Institute for Cancer Genetics and Informatics, Oslo 
 
Sponsor:  
Oxford University Hospitals NHS Foundation Trust 
 
Funder:  
Internal funding from Nuffield Division of Clinical and Laboratory 
Sciences, Oxford and Institute for Cancer Genetics and Informatics, Oslo 
£5000 award from Occtopus charity to cover sample retrieval costs 
Chief Investigator Signature:  
 
The investigators declare no potential conflicts of interest. 
 
 
Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other than the 
Sponsor, the Investigator Team, host organisation, and members of the Research Ethics Committee, 
unless authorised to do so. 
 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
 Page 2 of 16 
TABLE OF CONTENTS 
 
1. 
KEY STUDY CONTACTS ........................................................................................................................... 3 
2. 
SYNOPSIS ............................................................................................................................................... 4 
3. 
ABBREVIATIONS ..................................................................................................................................... 4 
4. 
BACKGROUND AND RATIONALE ............................................................................................................ 5 
5. 
OBJECTIVES AND OUTCOME MEASURES ............................................................................................... 6 
6. 
STUDY DESIGN ....................................................................................................................................... 6 
7. 
PARTICIPANT IDENTIFICATION .............................................................................................................. 7 
7.1. 
Study Participants .......................................................................................................................... 7 
7.2. 
Inclusion Criteria ............................................................................................................................ 7 
7.3. 
Exclusion Criteria ........................................................................................................................... 7 
8. 
STUDY PROCEDURES ............................................................................................................................. 8 
8.1. 
Recruitment ................................................................................................................................... 8 
8.2. 
Screening and Eligibility Assessment ............................................................................................. 8 
8.3. 
Informed Consent .......................................................................................................................... 9 
8.4. 
Study Visit ...................................................................................................................................... 9 
8.5. 
Data Handling ................................................................................................................................ 9 
8.6. 
Sample Handling .......................................................................................................................... 10 
8.7. 
Discontinuation/Withdrawal of Participants from the Study ..................................................... 10 
8.8. 
Definition of End of Study ........................................................................................................... 10 
9. 
STATISTICS ........................................................................................................................................... 10 
9.1. 
Description of Statistical Methods .............................................................................................. 11 
9.2. 
The Number of Participants ........................................................................................................ 11 
9.3. 
Analysis of Outcome Measures ................................................................................................... 11 
9.4. 
The Level of Statistical Significance ............................................................................................. 11 
9.5. 
Procedure for Accounting for Missing, Unused, and Spurious Data. .......................................... 11 
9.6. 
Inclusion in Analysis ..................................................................................................................... 11 
9.7. 
Procedures for Reporting any Deviation(s) from the Original Statistical Plan ............................ 11 
10. 
DATA MANAGEMENT ...................................................................................................................... 12 
10.1. 
Source Data ............................................................................................................................. 12 
10.2. 
Access to Data ......................................................................................................................... 12 
10.3. 
Data Recording and Record Keeping ....................................................................................... 12 
11. 
QUALITY ASSURANCE PROCEDURES ............................................................................................... 13 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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12. 
ETHICAL AND REGULATORY CONSIDERATIONS ............................................................................... 13 
12.1. 
Declaration of Helsinki............................................................................................................. 13 
12.2. 
Guidelines for Good Clinical Practice ...................................................................................... 13 
12.3. 
Approvals ................................................................................................................................. 13 
12.4. 
Reporting ................................................................................................................................. 13 
12.5. 
Participant Confidentiality ....................................................................................................... 13 
12.6. 
Expenses and Benefits ............................................................................................................. 14 
12.7. 
Other Ethical Considerations ................................................................................................... 14 
13. 
FINANCE AND INSURANCE .............................................................................................................. 14 
13.1. 
Funding .................................................................................................................................... 14 
13.2. 
Insurance ................................................................................................................................. 14 
14. 
PUBLICATION POLICY ....................................................................................................................... 14 
15. 
REFERENCES .................................................................................................................................... 15 
16. 
APPENDIX A:  AMENDMENT HISTORY ............................................................................................. 16 
 
1. KEY STUDY CONTACTS 
Chief Investigator 
Chris Cunningham 
Oxford University Hospitals NHS trust 
Department of Colorectal Surgery 
Churchill Hospital 
Old Road 
Headington 
 
Oxford OX3 7LE 
Tel: 01865 235657 
Email:   chriscunningham@nhs.net  
Fax: 01865 235857 
 
Sponsor 
Ms Heather House 
R&D Lead 
Research and Development Department 
Joint Research Office 
Block 60, Churchill Hospital 
Headington 
Oxford OX3 7LE 
E-mail: ouh.sponsorship@nhs.net  
Fax: 01865 572242 
 
 
 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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2. SYNOPSIS 
Study Title 
An observational study to correlate the results of ploidy and stroma 
analysis with prognosis in early rectal cancer 
Internal ref. no. (or 
short title) 
Ploidy and stroma in early rectal cancer 
Study Design 
Observational study 
Study Participants 
Rectal cancer patients 
Planned Sample Size 
150 patients 
Follow up duration 
Minimum 18 months 
Planned Study Period 
2 years 
 
Objectives 
Outcome Measures 
Primary 
 
To compare results of [A] ploidy 
and [B] stroma analysis with 
outcome after surgery to remove 
early rectal cancer 
Correlation between [A] ploidy 
status and [B] tumour: stroma 
ratio with local recurrence of 
cancer 
Secondary 
 
To compare results of [A] ploidy 
and [B] stroma analysis with 
survival after surgery to remove 
early rectal cancer 
To assess predictive value of [A] 
ploidy and [B] stroma analysis for 
outcome after surgery to remove 
early rectal cancer 
 
To compare results of [A] ploidy 
and [B] stroma analysis with 
outcomes by use of adjuvant 
treatment 
 
Correlation between [A] ploidy and 
[B] stroma analysis with overall 
survival after TEM surgery to 
remove rectal cancer 
Contribution of [A] ploidy and [B] 
stroma analysis to a regression 
model for local recurrence after 
TEM surgery to remove rectal 
cancer 
Subgroup analysis comparing [A] 
ploidy and [B] stroma results with 
local recurrence and survival for 
patients who did and did not 
receive 
adjuvant 
chemo- 
or 
radiotherapy 
 
3. ABBREVIATIONS 
CAG 
Confidentiality Advisory Group 
CI 
Chief Investigator 
CRA 
Clinical Research Associate (Monitor) 
CRF 
Case Report Form 
CRO  
Contract Research Organisation 
GCP 
Good Clinical Practice 
GP 
General Practitioner 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IRB 
Independent Review Board 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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MRN 
Medical Records Number 
NHS 
National Health Service 
NRES 
National Research Ethics Service  
PI 
Principal Investigator 
PIS 
Patient Information Sheet 
R&D 
NHS Trust R&D Department 
REC 
Research Ethics Committee 
SDV 
Source Data Verification 
SOP 
Standard Operating Procedure 
TEM 
Transanal endoscopic microsurgery 
TMF 
Trial Master File 
 
4. BACKGROUND AND RATIONALE 
Early rectal cancer can be removed by minimally-invasive surgery, and the standard pathological 
assessment of the removed tumour gives valuable information about how advanced the tumour is. This 
information is very important in indicating whether the cancer is likely to recur, and therefore in advising 
the patient after surgery whether further treatment is advisable, and if not, what is the most appropriate 
follow-up regime. However there is still a lot of uncertainty in these predictions about recurrence of the 
cancer, and better tests are being sought. This study aims to look at two further pathology tests, ploidy 
and stroma ratio in the tumour, and compare these test results with outcome in patients who have had 
an early rectal cancer removed. This will allow us to assess whether these two tests can give greater 
accuracy in predicting outcome. If so, we would be better able to advise patients with early rectal cancer 
about their prognosis and further management. 
Routine histopathology analysis of a rectal cancer specimen removed at surgery includes assessment of 
tumour size, depth of invasion, vascular, lymphatic and perineural invasion, tumour involvement of 
resection margins and nodal involvement. This information is valuable in predicting outcome. For 
example, predicted rates of local recurrence at 36 months following local excision of rectal cancer by 
transanal endoscopic microsurgery (TEM) based on tumour size, depth of invasion and lymphatic 
invasion have been tabulated [1]. However such models are not perfect, and leave room for 
improvement. Ploidy and stroma ratio are two further tests which have shown some promise in 
predicting outcome.   
Ploidy refers to the number of sets of chromosomes in a cell nucleus. Most human cells are normally 
diploid, with two sets of 23 chromosomes. Abnormal tumour cells may have a different number of sets 
of chromosomes, or be aneuploid, having some replicated or deleted chromosomes. In general, 
aneuploidy in cancer cells is associated with a worse prognosis. An early study of DNA ploidy in rectal 
cancer using flow cytometry showed an independent but small predictive effective of aneuploidy on 
survival [2]. Technological advances now allow more accurate and detailed assessment of ploidy. The 
DNA ploidy status of tumour cells in early ovarian cancer has been found to predict which patients will 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
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Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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benefit from adjuvant chemotherapy after surgery to remove the ovarian tumour and is used routinely in 
some centres to aid in decision-making [3].   
Stroma ratio refers to the tumour: stroma ratio. A lower proportion of tumour cells or, conversely, a 
higher percentage of stroma, in a cancer tends to be associated with a poorer prognosis. This ratio has 
been found to be strongly associated with tumour growth and invasion in colorectal cancers, and to 
independently predict survival in patients undergoing surgery to removal colorectal tumours [4]. 
However previous studies have looked mainly at more advanced colon cancers, rather than early rectal 
cancers, and have used only cancer-related death as the endpoint, rather than looking at local 
recurrence and response to adjuvant treatments.   
 
5. OBJECTIVES AND OUTCOME MEASURES 
Objectives 
Outcome Measures  
Timepoint(s) 
of 
evaluation of this 
outcome measure 
(if applicable) 
Primary Objective 
To compare results of [A] ploidy and 
[B] stroma analysis with outcome 
after surgery to remove early rectal 
cancer 
 
 
Correlation between [A] ploidy status and 
[B] tumour: stroma ratio with local 
recurrence of cancer  
 
Time 
between 
surgery for rectal 
cancer and local 
recurrence 
Secondary Objectives 
To compare results of ploidy and 
stroma analysis with survival after 
surgery to remove early rectal 
cancer 
To assess predictive value of [A] 
ploidy and [B] stroma analysis for 
outcome after surgery to remove 
early rectal cancer 
To compare results of [A] ploidy and 
[B] stroma analysis with outcomes 
by use of adjuvant treatment 
 
Correlation between [A] ploidy and [B] 
stroma analysis with overall survival after 
TEM surgery to remove rectal cancer 
 
Contribution of [A] ploidy and [B] stroma 
analysis to a regression model for local 
recurrence after TEM surgery to remove 
rectal cancer 
 
Subgroup analysis comparing [A] ploidy 
and 
[B] 
stroma 
results 
with 
local 
recurrence and survival for patients who 
did and did not receive adjuvant chemo- or 
radiotherapy  
 
Time 
between 
surgery for rectal 
cancer and death 
 
6. STUDY DESIGN 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
 Page 7 of 16 
This is an observational study comparing results of two specialised pathology tests with outcome after 
surgical removal of early rectal cancer. No extra interventions will be carried out on patients.  
Over 170 patients have already had an early rectal cancer removed by minimally-invasive surgery 
(transanal endoscopic microsurgery or TEM) since 2007 in Oxford, and more operations take place each 
month. Data about the tumour and follow-up on these patients is routinely collected in the Oxford TEM 
database to ensure that patients receive the appropriate investigations and management after surgery, 
and that any recurrence of the tumour is promptly detected and treated.  
For patients who agree to take part in this study, or who consented to donation of tissue for research at 
the time of surgery, we will perform the extra pathology tests on the tissue which is stored in the Oxford 
pathology department as standard practice, and compare the test results with the outcome information 
contained in the TEM database.  
The extra pathology tests will be undertaken in a specialist department in Oslo, Norway. The study will 
involve sending a small section of the preserved tissue from each consenting patient to Norway for 
analysis. After analysis, any residual parts of the samples will be returned to the Oxford pathology 
department. 
 
7. PARTICIPANT IDENTIFICATION 
7.1. Study Participants 
Participants diagnosed with rectal cancer that is operable and patients who have already had rectal 
cancer removed by TEM surgery in Oxford since 2007.     
7.2. Inclusion Criteria 
• 
Participant is willing and able to give informed consent (in English) for participation in the study, 
or gave informed consent for donation of tissue for research at the time of surgery. 
• 
Male or Female, aged 18 years or above. 
• 
Diagnosed with operable rectal cancer.  
• 
Due to undergo, or has already undergone, TEM surgery to remove rectal cancer. 
• 
A useable tissue sample has already been, or will be, taken as part of routine surgery. 
7.3. Exclusion Criteria 
The participant may not enter the study if ANY of the following apply: 
• 
Age less than 18  
• 
Adults who are not able to give consent or who are deemed vulnerable. 
• 
Participants who do not have a useable tissue sample will be excluded. 
 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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8. STUDY PROCEDURES 
8.1. Recruitment 
All patients who have undergone TEM surgery to remove a rectal cancer since 2007 are eligible, as well 
as new patients having surgery over the study period. Data on these patients is recorded as standard 
practice in the Oxford TEM database. There will be different groups, according to the time of surgery and 
whether or not potential participants consented for tissue donation, requiring different identification 
and recruitment strategies.  
Already had surgery groups 
Screening:  A member of the care team will extract a list of patients who have had TEM surgery for rectal 
cancer since 2007 from the TEM database, identified by MRN (OUH medical records number) only. The 
care team member will then screen the casenotes for these patients to determine whether or not they 
consented to donation of tissue for research on the surgical consent form.    
• 
Group A: Those who already consented for tissue donation 
The majority of potential participants signed a donation of tissue for research statement at the time of 
surgery. If this statement has been agreed to, patients will be considered to have consented to 
participate in the study. The care team member will pass the list of MRNs (and linked anonymized data) 
for these patients to the researcher.  
• 
Groups B & C: Those who did not consent to tissue donation at the time of surgery 
Once these have been identified, the care team member will identify whether these patients are still 
undergoing follow-up at Oxford hospitals. For those who are undergoing follow-up (Group B) the 
member of the care team will provide the researcher with the MRN and date of the next follow-up 
appointment. For those who are no longer under follow-up (Group C), the member of the care team will 
post an information sheet and consent form to the potential participant.  
For Group B the researcher will contact the patient during their follow-up visit and give them an 
information sheet about the study. They will be invited to sign a consent form if they wish to participate. 
Group C will receive an information sheet and consent form through the post, and will be provided with 
a telephone number should they wish to discuss the study further.  
New patients who have not yet had surgery (Group D) 
New patients, who have not yet had surgery, will be identified at colorectal cancer multi-disciplinary 
meetings. This will involve reviewing the clinical, histological and radiological investigations with other 
surgeons, oncologists, radiologists and pathologists as part of the normal clinical review process. Patients 
who are deemed suitable for TEM surgery will be considered eligible. Identified potential participants will 
then be approached by an investigator, who is a member of their direct care team, during their routine 
pre-operative clinic visit. They will be provided with an information sheet and given time to ask 
questions. Patients agreeing to participate will be recruited on their subsequent visit for surgery. 
8.2. Screening and Eligibility Assessment 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
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Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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Not applicable. Participation will be sought from patients undergoing or who have undergone TEM 
surgery for rectal cancer. 
8.3. Informed Consent 
Group A: Those who already consented to the anonymous use of donated tissue and anonymised data – 
these patients will be considered to have consented to surgery and no further consent will be sought. 
Group B: Those who did not consent to tissue donation at the time of surgery and are still visiting Oxford 
hospitals for follow-up – the researcher will contact the patient during their next visit and give them an 
information sheet. The patient will be given time to read the sheet and ask any questions they wish. They 
will then be asked to sign a consent form if they wish to participate.  
Group C: Those who did not consent to tissue donation at the time of surgery and are not still visiting 
Oxford for follow-up – these potential participants will receive an information sheet and consent form 
through the post. They will be requested to sign and return the consent form by post if they wish to 
participate. The information sheet will contain a telephone number for the patient to call if they wish to 
discuss the study further  
Group D: New patients who have not yet had surgery – potential participants will be approached by an 
investigator, who is a member of their direct care team, during their routine pre-operative clinic visit. 
They will be provided with an information sheet and given time to ask questions. Patients agreeing to 
participate will be asked to sign a consent form at that time, or on their subsequent visit for surgery if 
they want more time to consider the study.  
Groups B, C & D: The participant must personally sign and date the latest approved version of the 
Informed Consent form before any study specific procedures are performed. 
Written and, if seen in person, verbal versions of the Participant Information and Informed Consent will 
be presented to the participants detailing no less than: the exact nature of the study; what it will involve 
for the participant and the implications and constraints of the protocol. It will be clearly stated that the 
participant is free to withdraw from the study at any time for any reason without prejudice to future 
care, without affecting their legal rights and with no obligation to give the reason for withdrawal. 
The participant will be allowed as much time as wished to consider the information, and the opportunity 
to question the Investigator, their GP or other independent parties to decide whether they will 
participate in the study. Written Informed Consent will then be obtained by means of participant dated 
signature and dated signature of the person who presented and obtained the Informed Consent. The 
person who obtained the consent must be suitably qualified and experienced, and have been authorised 
to do so by the Chief/Principal Investigator. A copy of the signed Informed Consent will be given to the 
participant. The original signed form will be retained at the study site. 
8.4. Study Visit 
No specific visit is required. Consent will be taken when the patient attends for surgery, or for patients 
who have previously had surgery, consent will be taken during a routine follow-up visit or by post.  
8.5. Data Handling 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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For Group A patients, who gave consent at the time of surgery, a member of the care team will extract 
the relevant data, including tumour characteristics, any (neo-)adjuvant treatment details and outcome 
information, identified only by MRN, from the TEM database. The care team member will then securely 
transfer this anonymised data, identifiable by MRN only, to the research team. The research team will 
then allocate each participant a study ID according to the recruitment log, and replace the MRN with the 
unique study ID. The transfer will be done using a password protected spreadsheet sent via encrypted 
email.  For all other patients, who sign a specific study consent form, the researcher will prepare the data 
extract from the TEM database, identified only by MRN, and then replace the MRN with a study ID. The 
researcher will securely provide a list of MRNs, with date of surgery, to the pathology department. The 
MRNs will be linked to a unique study ID in a separate recruitment log. 
8.6. Sample Handling 
A list of MRN (medical record numbers) and date of the relevant surgery for consenting patients will be 
provided to the histopathology department so that the relevant tissue samples can be identified. The 
study tissue samples will be extracted from the removed surgical specimens by a pathologist after the 
routine pathological assessment. These sections will be anonymised and transported to the Institute for 
Cancer Genetics and Informatics in Oslo for further specialist histological assessment to obtain DNA 
ploidy of the tumour cells and the tumour: stroma ratio. These samples will be identifiable by their study 
ID only.  Any residual parts of the sections will be returned to Oxford to be held in the cellular pathology 
department, in line with standard practice for diagnostic surgical specimens.  
The DNA Ploidy test is done on monolayers prepared from 50μm sections after microdissecting the 
tumour 
area 
(a 
video 
of 
the 
laboratory 
procedure 
is 
available 
at 
www.youtube.com/watch?v=ERq17Bi3nGI ). The stroma measurement is done on standard HE sections 
(3μm).    
8.7. Discontinuation/Withdrawal of Participants from the Study 
Each participant has the right to withdraw from the study at any time.   
No additional procedures or observations will continue to be required after withdrawal.  
Withdrawal from the study will result in the exclusion of the data for that participant from analysis if the 
patient withdraws their consent to inclusion of their data.  
Withdrawn participants will be replaced by further recruitment to the study. 
The reason for withdrawal will be recorded in the CRF. 
8.8. Definition of End of Study 
Receipt of the final analysis from Oslo of the last sample. 
 
9. STATISTICS 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
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Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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9.1. Description of Statistical Methods 
All data will be stored in an Excel worksheet and analysed using Excel and R (a statistical programme). 
The DNA ploidy results and tumour: stroma ratio will be correlated with local recurrence and survival. A 
subgroup analysis will additionally correlate ploidy and stroma with local recurrence and survival with 
and without adjuvant treatment.  
A regression model will be developed incorporating ploidy and tumour: stroma ratio, along with a 
number of other known prognostic factors that are routinely collected, to assess whether ploidy and 
stroma have independent prognostic value in determining length of survival free of local recurrence, and 
length of disease-free survival.   
9.2. The Number of Participants 
The study will recruit 150 participants, including both those who have already had surgery since 2007 
and new patients having surgery after the study commences. Similar studies using these techniques of 
ploidy and stroma analysis compared to outcome after colorectal, breast and ovarian cancer that have 
produced significant results have generally used between around 150 and 300 patients [ref 3-6]. We 
expect to be able to recruit 150 patients, from amongst both those who have already had surgery and 
new patients, within a reasonable time-frame of 18 months.  
9.3. Analysis of Outcome Measures 
Data from all participants, excluding any that withdraw consent, will be used in the analysis. The primary 
outcome measure is the correlation between [A] ploidy status and [B] tumour: stroma ratio with local 
recurrence of cancer. Secondary outcome measures are i) correlation between [A] ploidy and [B] stroma 
analysis with overall survival after TEM surgery to remove rectal cancer, ii) the contribution of [A] ploidy 
and [B] stroma analysis to a regression model for local recurrence after TEM surgery to remove rectal 
cancer, and iii) Subgroup analysis comparing [A] ploidy and [B] stroma results with local recurrence and 
survival for patients who did and did not receive adjuvant chemo- or radiotherapy 
9.4. The Level of Statistical Significance 
A 0.05 level of significance will be used.  
9.5. Procedure for Accounting for Missing, Unused, and Spurious Data. 
All available data will be used. Missing data will not be imputed. Spurious data will be re-checked, and if 
valid will be included in the analysis. 
9.6. Inclusion in Analysis 
All patients who consent to the study will be included in the analysis.   
9.7. Procedures for Reporting any Deviation(s) from the Original Statistical Plan 
Not applicable. The analysis involves only simple statistical measures of readily available data.  
 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
CONFIDENTIAL 
Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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10. DATA MANAGEMENT 
10.1. 
Source Data 
Source documents are where data are first recorded, and from which participants’ CRF data are 
obtained. These include, but are not limited to, hospital records (from which medical history and 
previous and concurrent medication may be summarised into the CRF), clinical and office charts, 
laboratory records, diaries, radiographs, and correspondence. 
CRF entries will be considered source data if the CRF is the site of the original recording (e.g. there is no 
other written or electronic record of data).  All documents will be stored safely in confidential conditions. 
On all study-specific documents, other than the signed consent, the participant will be referred to by the 
study participant number, not by name. 
10.2. 
Access to Data 
Direct access will be granted to authorised representatives from the Sponsor and host institution to 
permit study-related monitoring, audits and inspections. 
10.3. 
Data Recording and Record Keeping 
For Group A patients who consented at the time of surgery, a member of the care team will prepare a 
data extract for all participants from the TEM database, in the form of an Excel spreadsheet, and securely 
transfer this anonymised data, identifiable by MRN only, to the research team. The research team will 
then allocate each participant a study ID according to the recruitment log, and replace the MRN with the 
unique study ID. For all other patients, who sign a specific study consent form, the researcher will 
prepare the data extract from the TEM database, identified only by MRN, and then replace the MRN with 
a study ID. All subsequent study data will be entered into the Excel spreadsheet. Data from this study will 
be generated at the Oxford University Hospitals and Institute for Cancer Genetics and Informatics, Oslo. 
Data will be analysed at the Oxford University Hospitals by the research investigators. Personal data will 
be stored and accessed by the investigators for less than 3 months. Research data will be stored for 10 
years after the completion of the study.  
The participants will be identified by a unique study specific number in the study database. For those 
who previously consented for tissue donation the MRN will be linked to the unique study number in the 
recruitment log. All other participants who consent will also be assigned a unique study identifier linked 
to their personal information. The name and any other identifying detail will NOT be included in any 
study data electronic file. 
All electronic data will be password-protected and anonymised. All hard copy files and data will be kept 
in a locked cabinet within a locked office with restricted access. Access to the data will be restricted to 
study team members. The NHS Code of Confidentiality will be followed. This study will follow the 
sponsor organisation’s policy regarding data storage and the NHS Code of Confidentiality. Information 
derived from the study will be stored on a single, secure (password encryption) NHS computer. This will 
be positioned within a secure location in the hospital.  

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
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Date and version No:     21 October 2016 v1.1 
 
 
 
 
 
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Participants will be informed using patient information sheets of how data will be stored (only 
participants who did not sign a tissue donation form at the time of surgery will receive patient 
information sheets). The principal investigators will constantly review the security of research data files.  
 
11. QUALITY ASSURANCE PROCEDURES 
The study will be conducted in accordance with the current approved protocol, GCP, relevant regulations 
and standard operating procedures. 
The study may be monitored or audited in accordance with the current approved protocol, GCP, relevant 
regulations and standard operating procedures.  
 
12. ETHICAL AND REGULATORY CONSIDERATIONS 
12.1. 
Declaration of Helsinki 
The Investigator will ensure that this study is conducted in accordance with the principles of the 
Declaration of Helsinki.  
12.2. 
Guidelines for Good Clinical Practice 
The Investigator will ensure that this study is conducted in accordance with relevant regulations and with 
Good Clinical Practice. 
12.3. 
Approvals 
The protocol, informed consent form and participant information sheet will be submitted to an 
appropriate Research Ethics Committee (REC), the Confidentiality Advisory Group (CAG) and host 
institution(s) for written approval. 
The Investigator will submit and, where necessary, obtain approval from the above parties for all 
substantial amendments to the original approved documents. 
12.4. 
Reporting 
The CI shall submit once a year throughout the clinical study, or on request, an Annual Progress Report 
to the REC, host organisation and Sponsor.  In addition, an End of Study notification and final report will 
be submitted to the REC, host organisation and Sponsor. 
12.5. 
Participant Confidentiality 
The study staff will ensure that the participants’ anonymity is maintained.  The participants will be 
identified only by a participant ID number on all study documents and any electronic database.  All 
documents will be stored securely and only accessible by study staff and authorised personnel. The study 

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An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
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will comply with the Data Protection Act, which requires data to be anonymised as soon as it is practical 
to do so.  No participant will be identified in any publication of the results in any way. 
12.6. 
Expenses and Benefits 
No visits additional to normal care are anticipated, so there is no expectation of patients incurring any 
expenses.  
12.7. 
Other Ethical Considerations 
The majority of potential patients will have consented to tissue donation and anonymous use, and use of 
anonymous data for research at the time of surgery. In order to link the tissue and data, the tissue must 
be made identifiable. It is not feasible to contact this participant group for consent to link the data and 
tissue; as the surgical procedures date back to 2007, many potential participants may have moved from 
the Oxford area and some may be deceased. The data and sample will be linked using the hospital MRN 
number. This is a trust-specific reference number outside the OUHFT participants are not identifiable by 
this number. Once linked the data and samples will also be re-anonymised (or linked anonymised). There 
will be no onward disclosure of patient data outside the care team, as the researcher is also part of the 
participants’ care team.  
Tissue samples will be transferred outside the UK. A material transfer agreement will be in place to cover 
the terms of the transfer of the samples to the Institute of Cancer Genetics and Informatics in Oslo. The 
samples will be identifiable by study ID only, when they are transferred to Norway. Participants consent 
to the transfer of their linked anonymised samples to Norway. 
13. FINANCE AND INSURANCE 
13.1. 
Funding 
Internal funding from Nuffield Division of Clinical and Laboratory Sciences, Oxford will cover the costs of 
retrieving the pathology specimens, and from the Institute for Cancer Genetics and Informatics, Oslo will 
cover the processing and analysis of the specimens. 
13.2. 
Insurance 
NHS bodies are legally liable for the negligent acts and omissions of their employees. If you are harmed 
whilst taking part in a clinical study as a result of negligence on the part of a member of the study team 
this liability cover would apply. 
Non-negligent harm is not covered by the NHS indemnity scheme. The Oxford University NHS Trust, 
therefore, cannot agree in advance to pay compensation in these circumstances. 
In exceptional circumstances an ex-gratia payment may be offered. 
 
14. PUBLICATION POLICY 

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An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
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REC reference: 16/WM/0443 
 
 
 
 
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The Investigators will be involved in reviewing drafts of the manuscripts, abstracts, press releases and 
any other publications arising from the study.  Authors will acknowledge the source of funding for the 
study. Authorship will be determined in accordance with the ICMJE guidelines and all other contributors 
will be acknowledged. 
15. REFERENCES 
1. Bach SP, Hill J, Monson JR, Simson JN, Lane L, Merrie A, et al. A predictive model for local recurrence 
after transanal endoscopic microsurgery for rectal cancer. The British Journal of Surgery. 2009;96(3):280-
90  
2. Goh HS, Jass JR, Atkin WS, Cuzick J, Northover JMA. Value of flow cytometric determination of ploidy 
as a guide to prognosis in operable rectal cancer: a multivariate analysis. Int J Colorectal Dis. 
1987;2(1):17-21 
3. Kristensen GB, Kildal W, Abeler VM, Kaern J, Vergote I, Tropé CG, Danielsen HE. Large-scale genomic 
instability predicts long-term outcome for women with invasive stage I ovarian cancer. Ann Oncol (2003) 
14 (10): 1494-1500 
4. Park JH, Richards CH, McMillan DC, Horgan PG, Roxburgh CSD. The relationship between tumour 
stroma percentage, the tumour microenvironment and survival in patients with primary operable 
colorectal cancer, Ann Oncol (2014) 25 (3): 644-651. 
5. Downey CL, Simpkins SA, White J et al. The prognostic significance of tumour-stroma ratio in 
oestrogen receptor-positive breast cancer. Br J Cancer. 2014;110:1744-1747. 
6. West NP, Dattani M, McShane P et al. The proportion of tumour cells is an independent predictor for 
survival in colorectal cancer patients. Br J Cancer. 2010;102(10):1519-1523. 
 

Date and version No:     21 October 2016 v1.1 
An observational study to correlate the results of ploidy and stroma analysis with prognosis in early rectal cancer 
IRAS reference: 195159 
REC reference: 16/WM/0443 
 
 
 
 
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16. APPENDIX A:  AMENDMENT HISTORY 
 
Amendment 
No. 
Protocol 
Version 
No. 
Date issued 
Author(s) 
of 
changes 
Details of Changes made 
 
 
 
 
 
 
Protocol amendments must be submitted to the Sponsor for approval prior to submission to the REC 
committee.