Characterising Biomarkers and Clinical Algorithms to Identify Oncology Patients on Immune Checkpoint Inhibitors (ICPI) That Are at Greater Risk of Developing Immune-related Adverse Events (irAE)
Conditions: Neoplasms
Sponsor: Portsmouth Hospitals NHS Trust

 
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Qualitative Protocol Development Tool 
 
Full/Long Title of Trial 
Characterising biomarkers and clinical algorithms to identify oncology patients on immune checkpoint 
inhibitors (ICPI) that are at greater risk of developing immune-related adverse events (irAE). 
 
Short Trial title/ Acronym 
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Protocol version number and date 
Version 1.3, 2nd January 2024 
RESEARCH REFERENCE NUMBERS 
 
IRAS Number: 
329576 
 
SPONSORS Number: 
Not applicable 
 
FUNDERS Number: 
Not applicable 
 
 
 

 
 
 
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SIGNATURE PAGE 
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 in the Declaration of Helsinki, the Sponsor’s SOPs, and other regulatory requirement. 
I agree 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. 
I also confirm that I will make the findings of the study 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. 
 
For and on behalf of the Study Sponsor: 
Signature:  
...................................................................................................... 
 
Date: 
....../....../...... 
Name (please print): 
...................................................................................................... 
 
 
Position: 
...................................................................................................... 
 
 
 
Chief Investigator: 
Signature: 
...................................................................................................... 
 
Date: 
....../....../...... 
Name: (please print): 
...................................................................................................... 
 
 
 
 
 
 
 
 
 
 

 
 
 
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LIST of CONTENTS 
GENERAL INFORMATION 
Page No. 
TITLE PAGE 
 
i 
SIGNATURE PAGE 
ii 
LIST OF CONTENTS 
 
iii 
KEY STUDY CONTACTS 
iv 
STUDY SUMMARY 
vi 
FUNDING 
 
vi 
ROLE OF SPONSOR AND FUNDER 
vii 
ROLES & RESPONSIBILITIES OF STUDY STEERING GROUPS AND INDIVIDUALS  
 
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STUDY FLOW CHART  
viii 
STUDY PROTOCOL 
1. LAY SUMMARY 
9 
2. BACKGROUND 
10 
3. OBJECTIVES AND OUTCOME MEASURES/ENDPOINTS 
11 
4. STUDY DESIGN  
12 
5. SAMPLE PROCESSING  
14 
6. DATA PROCESSING  
15 
7. SAMPLE SIZE 
16 
8. STUDY MANAGEMENT  
16 
9. HEALTH RELATED FINDINGS 
16 
10. END OF STUDY 
16 
11. FUNDING 
16 
12. IMPORTANCE AND POTENTIAL BENEFIT 
17 
13. PUBLIC AND PATIENT INVOLVEMENT  
17 
14. DISSEMINATION/ IMPLEMENTATION OF RESEARCH 
18 
 

 
 
 
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KEY STUDY CONTACTS 
Chief Investigator 
Dr Laura Wainwright 
Queen Alexandra Hospital 
Southwick Road 
Cosham 
PO6 3LY 
laura.wainwright@porthosp.nhs.uk 
023 9228 6345 
Lead Researcher 
Mrs Louise E Duvall 
Queen Alexandra Hospital 
Southwick Road 
Cosham 
PO6 3LY 
louise.duvall@porthosp.nhs.uk 
023 9228 4845 
Sponsor 
Mr Joe Shoebridge 
Research and Development Department 
Queen Alexandra Hospital 
Southwick Road 
Cosham 
PO6 3LY 
research.office@porthosp.nhs.uk 
023 9228 6236 
Funder(s) 
National School of Healthcare Science 

 
 
 
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213 Hagley Road 
Birmingham 
B16 9RG 
0121 695 2529 
nshcs@hee.nhs.uk 
 
 
 
 

 
 
 
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STUDY SUMMARY 
Study Title 
Characterising biomarkers and clinical algorithms to identify 
oncology patients on immune checkpoint inhibitors (ICPI) that are 
at greater risk of developing immune-related adverse events (irAE).  
Internal ref. no. (or short title) 
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Study Design 
Retrospective cohort study 
Study Participants 
 Adult oncology patients at Queen Alexandra hospital, who are 
prescribed FDA approved ICPI for the first time 
Planned Size of Sample (if applicable) 
120 
Follow up duration (if applicable) 
6 months post initiation of ICPI 
Planned Study Period 
Sample collection = 18 months 
Data collation and analysis = 6 months 
Total time = 24 months 
Research Question/Aim(s) 
 
Can a clinical algorithm be created to flag high risk Oncology 
patients that have a greater risk of developing irAE? In addition, 
can novel biomarkers such as malondialdehyde be incorporated 
into this algorithm to aid stratification. 
 
FUNDING AND SUPPORT IN KIND 
FUNDER(S) 
FINANCIAL AND NON-FINANCIAL SUPPORT 
GIVEN 
National School of Healthcare Science 
 
£13,000 per year for 5 years 
£65,000 total 
Royal College of Pathologists 
Awarded £1015.60 2nd of August 2023 
 
 
 
 

 
 
 
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ROLE OF STUDY SPONSOR AND FUNDER 
Queen Alexandra Hospital is the sponsor of this research study. However, the responsibility for the design, 
conduct, data analysis, interpretation, manuscript writing, and dissemination of results falls to the lead 
researcher Mrs Louise E Duvall. The sponsor will have oversight of the study and ensure that it continues to 
operate within legal boundaries. The chief investigator and lead researcher are responsible for ensuring that 
the sponsor is kept informed of changes and updated to the BIO-CHECKPOINT study.  
Funder(s) do not control the final decision regarding any of these aspects of the study.  
 
ROLES AND RESPONSIBILITIES OF STUDY MANAGEMENT COMMITEES/GROUPS & INDIVIDUALS 
Study Steering Groups 
No study steering groups have been involved in the study coordination and conduct. 
Patient and Public Involvement group has been engaged at Portsmouth and research study will be presented to 
this group. Public presentation will also be given at Manchester May 2024 as part of Louise E Duvall’s 
Doctorate in Clinical Biochemistry. 
 
 
PROTOCOL CONTRIBUTORS 
The study design is principally the responsibility of the lead researcher Louise E Duvall and Chief investigator 
Laura Wainwright. The funder the National School of Healthcare Science does not control any aspect of the 
study. The sponsor the Research and Development office at Portsmouth will review and feedback on the 
protocol when indicated. The study design will be discussed and review with the Portsmouth Patient 
involvement group, facilitated by Sharon Court.  
 
KEY WORDS: 
Check point inhibitors, oncology, cancer, immune related 
adverse events, blood biomarkers  
 
 
 

 
 
 
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STUDY FLOW CHART 
 
Write up and communication of results
Study concludes patient results correlated to clinical outcomes
Repeat process depending on how many cycles patient is prescribed
Samples destroyed in line with human tissue act, 2004 and local pathology protocols
Stored serum/plasma tested in batches once a month
Store left over clinic plasma/serum
Patient has repeat bloods 4-6 weeks post intial treatment
Samples destroyed in line with human tissue act, 2004 and local pathology protocols.
Stored serum/plasma tested in batches once a month
Booked in under pseudoname/ reference
Baseline clinic bloods taken prior to treatment. Left over serum and plasma stored
Patient identified by study by chemo scheduling system
Clinical decision to treat with FDA approved ICPI
Patient diagnosed with cancer referred to Oncologist

 
 
 
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STUDY PROTOCOL 
1.0           LAY SUMMARY –  
Immune checkpoint inhibitors (ICPI) are a type of cancer treatment. Unlike traditional chemotherapy 
and radiation, which damages both the cancer and the healthy tissue, ICPI targets cancer directly by 
altering the immune system. Cancer cells produce high levels of protein called checkpoint proteins, 
which bind to white blood cells (which are part of the immune system) and stops them from 
working. Effectively the cancer is pushing a stop button on the immune system and the body can no 
longer fight it off.  ICPIs block and remove these cancer checkpoint proteins, which allows the 
immune system to target the cancer again removing this stop button.   
ICPI has great success in treating cancer and 10% of oncology NHS patients receive this treatment, 
although this number is increasing. However, ICPI carries a risk of a type of side effect called immune 
related adverse events (irAE). irAEs can be life threatening and present with similar symptoms to the 
patient’s cancer. For example, a patient may have kidney cancer and after treatment with ICPI 
develop kidney failure. It is difficult for the doctor to tell if this is the cancer progressing or a side 
effect of the treatment. Delays in diagnosing irAE can lead to unnecessary hospitalisation, 
unnecessary breaks in treatment, lifelong side-effects, and death. 
Currently there is not a unified blood test panel for ICPI patients, and the cancer societies have 
produced little guidance for the doctors to use. At Portsmouth what blood tests you get as an ICPI 
patient depend on which clinician you see. There is also little research as researchers are focussing 
on using blood tests to predict ICPI treatment success rather than the chance of a patient developing 
an irAE. 
We intend to conduct a study where we collect leftover blood from routine clinical blood draws on 
oncology patients being treated with ICPIs for the first time. We will freeze the leftover samples and 
test them a month later for different routine blood tests as well as malondialdehyde. 
Malondialdehyde is a blood test that can assess the body’s oxidative stress level, a condition where 
your body lacks antioxidants. Testing for malondialdehyde is not available in the NHS and we would 
produce a new NHS blood test as part of this study. Once testing is completed the samples will be 
destroyed as per our normal protocol. At the end of the study, we will correlate the blood test 
results to the patient’s outcome i.e., did they have an irAE or not and assess if there are any 
differences in their blood test results. From this information we hope to understand which blood 
tests help to highlight if a patient is at a risk of developing irAE before it occurs. It also aims to 
develop a new method for measuring malondialdehyde.  
 

 
 
 
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2.0 
BACKGROUND 
This study aims to identify biomarkers that stratify Oncology patients on immune check point 
inhibitor (ICPI) that have an increased risk of developing autoimmune toxicity known as immune-
related adverse events (irAE). 
ICPIs are being increasingly prescribed and 10% of NHS Oncology patients receive treatment from 
this drug group (1,2).  The reported rate of total irAE triggered by ICPI varies but is thought to be 
between 40-66% (3–5), a third of which are endocrine related (6,7). 
Several bodies including the European Society for Medical Oncology, American Society for Clinical 
Oncology and the National Comprehensive Cancer Network have produced guidance for irAE 
management, but no unified screening protocol exists to stratify patients at high-risk (8). Currently 
the literature primarily centres around looking for prognostic biomarkers for treatment success 
rather than characterising toxicity.  
Individual studies review the utilisation of some routine clinical blood tests, i.e., albumin, 
rheumatoid factor, lactate dehydrogenase, IL-6, etc, to screen and stratify high-risk patients (9,10).  
However, these tests are used in isolation and no singular clinical algorithm using a variety of routine 
tests has been developed for initial screening during ICPI therapy. A variety of protocols have been 
developed for screening endocrine irAE, although these are not consistently adopted, and practice 
varies.  
ICPIs induce an enhanced immunological response to promote anti-tumour activity (11). There are a 
few forms of ICPI including CTLA-4 antibodies that upregulate T-cell activation pathways to enhance 
the tumour immune response. There are also antibodies that act as programmed death receptor 
agonists, which promote cytokine release and T-cell activation. (12) 
An array of studies has shown that reactive oxygen species (ROS) are involved in the regulation of T-
cell anti-tumour immunity (13–20). ICPI are active therapies that alter T-cell intracellular signalling, 
which can increase ROS production and proinflammatory cytokine secretion, thus exerting an 
immunotherapeutic effect (13).  
irAE refers to a spectrum of autoimmune toxicities triggered by ICPI. ROS are implicated in the 
initiation, amplification, pathogenesis of autoimmune diseases (21,22). Are ROS heavily upregulated 
in irAE due to a dysregulated immune response and could the measurement of an oxidative stress 
marker act as a marker of toxicity? 
Malondialdehyde is generated from ROS driven lipid peroxidation of polyunsaturated fatty acids and 
is an indirect marker of oxidative stress. Malondialdehyde can be detected within plasma, serum and 
urine via liquid-chromatography mass-spectrometry (LC-MS/MS). (23–25) Malondialdehyde has 
been suggested as a future marker of tissue level thyroid dysfunction, the most common irAE is 
hypothyroidism (26).  

 
 
 
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At present, Portsmouth does not have a unified screening process for patients about to commence 
ICPI and tests requested are clinician dependent. This project seeks to produce a unified clinical 
algorithm. 
3.0 Objectives and Outcomes Measures/Endpoints 
3.1 Primary objectives  
Can a clinical algorithm be created to flag high risk Oncology patients that have a greater risk of 
developing irAE? In addition, can novel biomarkers such as malondialdehyde be incorporated into 
this algorithm to aid stratification. 
 
 
P 
Oncology adult patients on ICPI 
 
I 
No intervention – retrospective analysis  
 
C 
Comparing blood test results of those who develop irAE to those who don’t 
 
O 
Are any of the blood tests significantly different in those who develop irAE 
 
T 
6 months follow up post starting treatment, 18 testing period.  
 
3.2 Secondary objectives  
This principal research question will be broken down to the following sub aims:  
 
1. Are there any established biochemistry, haematology and immunology blood tests that produce 
significantly different results in those who develop irAE when compared to those that don’t. 
Including IL-6 (requires in-house verification). Observational study 
 
2. Can a malondialdehyde LC-MS/MS assay suitable for clinical laboratory use be developed based 
on a method development by Sobsey et al. 
 
3. Is malondialdehyde significantly raised in those who develop irAEs compared to those that do not. 
An ELISA kit has been located in the event an LC-MS/MS method is not successfully developed. 
 
4. Can a blood test clinical algorithm be developed to stratify patients at high-risk of developing an 
irAE. 
 
3.3 Outcome 
This project could produce a clinical algorithm and/or blood test panel that successfully highlights 
patients at a high risk of developing irAE, prior to commencing therapy. This would allow closer 
monitoring of high-risk patients, leading to earlier diagnosis of irAE. Currently irAE can be tricky to 
diagnose and have overlapping symptoms with the malignancy and related concomitant treatments 
(27). Earlier diagnosis would lead to reduced hospitalisation, better outcomes, and reduced 

 
 
 
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interruptions to the anti-cancer therapy, which may lead to better malignancy curative rates. There 
is also a potential to reduce morbidity and mortality risk for this subset of patients, as well as an 
overall reduction in resource burden. 
4.0 STUDY DESIGN  
4.1 SUMMARY OF DESIGN 
This is a retrospective cohort study that looks at blood test results correlated to clinical outcomes. 
Oncology patients, whom have been prescribed ICPIs for the first time, will be identified via the 
chemotherapy scheduling system. This study does not require extra blood draws or tubes to be 
taken. Left over serum and plasma from routine clinical blood draws will be retrieved. This serum 
and plasma will be aliquoted into tube labelled with a pseudonym reference. The samples will be 
booked into our laboratory information management system under a pseudonym reference. This 
system is password protected and only those involved in this research should be accessing these 
records. Left-over samples will be frozen at -80C and stored for a period of 1 month before being 
tested in batches. Before testing a list of NHS numbers of the patients that have been recruited will 
be sent to the Trust’s data office. All samples for patients that have opted out of having their records 
used for research purposes will be destroyed in line with local pathology policy. Once testing is 
completed the left-over samples will be disposed of as in line with the Human Tissue Act, 2004 and 
local Portsmouth Pathology protocols.  
Louise E Duvall lead researcher will correlate the results to the patients’ identifiers in a password 
protected sheet. Results will then be clinically correlated to patient’s records to deem patient 
outcomes and if they had an irAE. Data will be correlated into a password protected excel sheet kept 
on Trust computer system. Data will then be de-identified and patient identification will be 
removed. Identifiable patient data will be removed for data analysis the spread sheet would contain 
age in years, sex, cancer type, ICPI type, if they had an irAE, irAE type and when it occurred. This data 
will be in a password protected excel sheet. Statistics will be carried out via R.  
Minimal patient data will be used in the write up of this study i.e., age in years, sex, cancer type, ICPI 
treatment type, and if they had an irAE. Information about irAE will include type of event, and when 
it occurred post treatment.   
4.2 ELIGIBILITY CRITERIA  
Inclusion criteria  
• 
≥18 years of age 
• 
All sex 
• 
Oncology patients 
• 
Who is prescribed FDA approved check point inhibitors: (ipilimumab, nivolumab, 
pembrolizumab, cemiplimab, atezolizumab, avelumab, and durvalumab) 

 
 
 
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• 
All cancer subtypes are included 
Exclusion criteria  
• 
<18 years of age 
• 
Those previously been treated with checkpoint inhibitors. 
• 
Those with a previous medical history of autoimmune disease 
• 
Those with previous medical history of endocrine diseases 
• 
Those with a pre-existing malignancy  
• 
Non-Queen Alexandra hospital oncology patients  
• 
Lacking capacity to consent 
 
4.3 ASSESSMENT AND MANAGEMENT OF RISK 
This is a low risk study, and is categorised as a study limited to working with human tissue samples 
and/or analysis of data. There will be no research specific participant contact or observation and 
therefore no AEs/SAEs would occur because of study participation.  
4.4 STUDY PROCEDURES 
Please refer to the flow chart on page viii for a summary of the study procedures. The study 
procedures described below will be conducted in accordance with SOPs and/or work instructions 
provided by the CI. 
4.5 STUDY INTRODUCTION 
This study seeks to gain consent from patients remotely via the telephone. Samples will be 
scavenged from routine care samples (no additional procedures will occur i.e. blood draws etc.). The 
study is being designed to limit the burden on clinical staff. The NHS is currently facing massive 
pressures and unprecedented demand. However, we should continue to seek improvement this 
study has been designed so we can continue to improve our services while putting minimum burden 
on the Oncology staff. This project is part of a service improvement project to highlight which blood 
tests are best at predicting irAEs. 
4.6 STUDY OPT OUT 
Patients that have been recruited on the study will have their NHS numbers extracted into a list. 
Once a month before running of the sample batches, this list of NHS numbers will be raised on the 
Trust’s internal system called Mycall http://MyCall as a National Data Opt out.   Any patients who 
have opted out of having their data and records used for research will be removed from the study 

 
 
 
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and the samples destroyed as in line with local pathology procedures. This is in line with local trust 
policy that has been created to ensure compliance with the NHS National Data Opt-out policy. 
4.7 STAFF TRAINING 
Information about the study training will be provided by the research team. Local in house training 
procedures will be followed for the operation of the analysers. Louise E Duvall will receive additional 
research based training as part of her Doctorate with the University of Manchester. Louise E Duvall 
has also completed Good Clinical Practice basic research training. 
 
5.0 SAMPLE PROCESSING 
5.1 SAMPLE COLLECTION AND STORAGE 
Patients will be identified via the chemotherapy scheduling system, which will be reviewed and 
checked by lead researcher Louise Duvall daily Monday-Friday.  Patients who are yet to start ICPI will 
be listed in a password protected sheet with the expected day of their chemo. The lead researcher 
will then check to assess that if the patients listed meet the inclusion criteria and if they have any of 
the features of the exclusion criteria. Those that meet the exclusion criteria will be deleted off the 
list. The laboratory information management system will be reviewed daily to assess if we have 
received their clinical bloods. Left over serum and plasma will be stored frozen and tested in batches 
once a month.  
5.2 PATIENT CONSENT 
Enrolment onto this study required patient consent; this will be achieved by telephone. Samples will 
not be tested unless patient consent has been obtained.  Once eligible patients have been selected 
and their samples have been stored a member of the research team will contact the patient as per 
BIO-CHECKPOINT Telephone Consent Script, version 1.0. The research team will offer to send the 
patient the PLS BIO-CHECKPOINT adults version 1.0 information leaflet and arrange to call back at a 
suitable time to take consent recorded via the BIO-CHECKPOINT version 1.0 Consent Form. 
Participants expressing an interest in the study will have their eligibility assessed, receive a PIS and 
will have adequate time to read this information. The information will also be presented to them 
verbally to ensure they understand. If they would like to, they will be able to talk to relevant health 
care professionals or family members about involvement in the study and will have the opportunity 
to ask any questions they may have before consent is taken.  If the patient does not consent to the 
study the samples will be destroyed and not tested.  
 

 
 
 
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5.3 SAMPLE ANALYSIS  
Residual serum and plasma that will be anonymised will be defrosted and run at the Blood Sciences 
laboratory in Portsmouth. 
Blood tests will be run on UKAS ISO15189 accredited blood analysed by stated registered Clinical 
Scientists and Biomedical Scientists. Malondialdehyde levels will be assessed via isotope dilution 
mass spectrometry method or an ELISA kit in the event of method validation failure. The method will 
be fully validated and installed at Portsmouth Pathology laboratories. It is not anticipated that there 
will be any sample remaining at the end of the analysis procedure. Any sample that remains at the 
end of the analysis procedure will be destroyed as per standard NHS Pathology Department 
guidelines. 
5.4 REPORTING OF ACTIONABLE RESULTS 
Critically abnormal results may be produced during this study. To avoid moral dilemma of if to report 
results that the clinician did not request the following mitigating factors have been put into place. 
Samples will be labelled and booked in under a pseudonym and only the lead researcher will have 
access to the key and relevant patient identifiers. Samples will be batched and run once a post 
collection, making these results historic values. Results will not be clinically correlated to patient 
outcomes until May 2025 meaning outcomes of any abnormal results would have likely presented by 
this point. Clinicians will not see or be able to look up the study’s results.  
6. DATA PROCESSING 
6.1 DATA TRANSFER AND STORAGE 
Results will be anonymised under a pseudonym/reference on the laboratory information 
management system (APEX). This is a password protected system that only authorised personnel can 
access on password protected Trust accounts, which are also password protected. The patients’ 
clinicians will not be able to see these results or link them to their patients. Only those on the 
research team will be able to de-anonymise the data. Results will be stored on APEX during the 
study. Post study completion the results will be extracted from APEX into a password protected 
excel sheet. Results and patient identifying data will not be sent outside of the Trust. 
6.2 SOURCE DATA 
Laboratory results, CVs, delegation logs will be deemed source data. 
6.3 DATA CONFIDENTALITY  

 
 
 
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The central research team Portsmouth Pathology i.e., Laura Wainwright and Louise Duvall will have 
access to patient data and anonymised result data. This data will be stored securely on password 
protected computers at the NHS Blood Sciences laboratory in Portsmouth. Patient identifiable data 
will not be stored outside of the Trust computers. Linkage of test results to personal identifiable 
information will only be possible through the pseudoname/reference. Linkage will only be carried 
out by Louise E Duvall.  Statistics will be carried out by Louise E Duvall as this study is part of their 
Doctorate in Clinical Science awarded by the University of Manchester. Statistics will be carried out 
using R on a password protected personal computer. Data for statistics will not contain patient 
identifiable data i.e., first name, surname, date of birth, hospital number, NHS number, post code, 
etc.  Data will be stored for 1 year post completion of the study before being archived or deleted in 
line with the sponsors’ policy.  
6.4 DATA ARCHIVING  
At the end of the study, archiving will be carried out in accordance with sponsor protocols and, 
where relevant, local site governance requirements. 
7.0 SAMPLE SIZE  
All adult oncology patients at Queen Alexandra Hospital who are prescribed FDA approved ICPI for 
the first time, providing they don’t meet the exclusion criteria will be recruited. The sample size is 
expected to be around 120 patients, based on ICPI patient Portsmouth Hospital University Trust data 
from 2022. It is estimated that around 40-66% of patients on ICPI will have an irAE based on the 
literature. 
Assuming an incidence rate of 40% for irAE, this study would require at least a sample size of 47 
individuals assuming a significance criterion of α=0.05 and power =80. 
The study will run for 18 months and hopes to obtain at least a sample size of 47 individuals, depending 
on how many patients are referred for ICPI therapy. The study will end before 18 months if we recruit 
150 patients before the end date. 
 
The period of testing was decided upon discussion with the Oncology department, based on ICPI 
prescriptions provided by the pharmacy department.  
8.0 STUDY MANAGEMENT 
The chief investigator and lead researcher are responsible for the day-to-day operation of the study. 
Management support for this study will be provided by the Research and development department 
at Portsmouth Hospital University Trust.  

 
 
 
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9.0 HEALTH RELATED FINDINGS 
Samples will be stored and tested under a pseudonym/reference one month post initial collection. 
Clinicians will not have access to these results and those testing the samples will not know who the 
sample belongs too. Results will not be linked to the patients’ records until completion of the data 
collection period i.e., May 2025.    
10.0 END OF STUDY 
The study will finish 6 months post the final analysis of samples has been undertaken. 
11.0 FUNDING 
This study will be funded by the Higher Specialist Scientist Training (HSST) program via the National 
School of Healthcare Science and by the Royal College of Pathologists Fellowship Research Start-up 
Grant.  
12.0 MONITORING AND INDEMNITY 
Monitoring of this study will ensure compliance with Good Clinical Practice. The Investigator (s) will 
permit monitoring, audits, REC review and regulatory inspections by providing the Sponsor, 
Regulates and REC direct access to source data and other appropriate documents (e.g., study data 
collection forms).  
NHS Indemnity will apply.  
13.0 IMPORTANCE AND POTENTIAL BENEFIT 
This study is aiming to act as service improvement collaboration between Oncology and Pathology 
services at Portsmouth Hospital University trust. To help highlight which blood tests help to highlight 
ICPI at risk of developing irAE.  On the basis of the results produced we aim to produce a blood test 
panel that can be ordered on our blood requesting software (ICE) for all ICPI patients. This will help 
to increase consistency in blood test requesting and diagnosis of ICPI irAE faster. 
This study will also further the progression of Louise E Duvall’s career and professional development. 
This will allow development of mass-spectrometry skills, data processing and analysis as well as 
journal publications. Completion of this project will also feed in to Louise E Duvall’s Royal College of 
Pathologists exams for her fellowship.  

 
 
 
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14.0 PUBLIC AND PATIENT INVOLVEMENT  
Research was presented to research team at Portsmouth Hospital University trust design was 
enthusiastically received.  
Sharon Court patient public involvement coordinator has been approached; research will be 
discussed at an upcoming meeting including the acceptability, design, management, undertaking, 
and analysis of results.  
Results will also be presented to the Portsmouth Patient Involvement Group.  Research will also be 
presented to patient representatives as part of Louise E Duvall’s doctorate in Clinical Science with 
the University of Manchester.  
15.0 DISSEMINATION/ IMPLEMENTATION OF RESEARCH 
Results will be written up and submitted for publication in a peer-reviewed journal. Abstracts will be 
submitted to national conferences. Results will be presented to clinical colleagues at regular in-
house meetings. Results will also be presented to the Portsmouth Patient Involvement Group. This 
study will be submitted as part of a Doctorate in Clinical Science (DClinSci) with the University of 
Manchester and as part of Fellowship exams with the Royal College of Pathologists for the student 
Louise E Duvall.  
16.0 REFERENCES 
1. Robert C. A decade of immune-checkpoint inhibitors in cancer therapy. Nat Commun. 2020 Jul 
30;11(1):3801.  
2. Guinan TJ, Garbutt N, Olsson-Brown A. The impact of an immuno-oncology service at a regional 
cancer centre based in the north west of the UK. Br J Nurs. 2022 Apr 21;31(8):414–20.  
3. Jennings S, Anstey S, Bower J, Brewster A, Buckman J, Fenlon D, et al. Experiences of cancer 
immunotherapy with immune checkpoint inhibitors (ExCIm)—insights of people affected by 
cancer and healthcare professionals: a qualitative study protocol. BMJ Open. 2021 May 
1;11(5):e043750.  
4. Ellenger K, Flatley M, Spencer K, Clarke K, Franks K, Jain P. P2.04-21 Real World Experience of 
Immune Checkpoint Inhibitors In NSCLC: Our First 10 Months Experience at Leeds Cancer 
Centre, UK. Journal of Thoracic Oncology. 2018 Oct 1;13(10):S738.  
5. Khair DO, Bax HJ, Mele S, Crescioli S, Pellizzari G, Khiabany A, et al. Combining Immune 
Checkpoint Inhibitors: Established and Emerging Targets and Strategies to Improve Outcomes in 

 
 
 
BIO-CHECKPOINT 
 
   
 
19 
Final Version 1.2 12th September 2023-BIOCHECKPOINT 
 
Melanoma. Frontiers in Immunology [Internet]. 2019 [cited 2023 Jan 11];10. Available from: 
https://www.frontiersin.org/articles/10.3389/fimmu.2019.00453 
6. Villa NM, Farahmand A, Du L, Yeh MW, Smooke-Praw S, Ribas A, et al. Endocrinopathies with 
use of cancer immunotherapies. Clinical Endocrinology. 2018;88(2):327–32.  
7. Husebye ES, Castinetti F, Criseno S, Curigliano G, Decallonne B, Fleseriu M, et al. Endocrine-
related adverse conditions in patients receiving immune checkpoint inhibition: an ESE clinical 
practice guideline. European Journal of Endocrinology. 2022 Dec 1;187(6):G1–21.  
8. Brahmer JR, Abu-Sbeih H, Ascierto PA, Brufsky J, Cappelli LC, Cortazar FB, et al. Society for 
Immunotherapy of Cancer (SITC) clinical practice guideline on immune checkpoint inhibitor-
related adverse events. J Immunother Cancer. 2021 Jun 1;9(6):e002435.  
9. Michailidou D, Khaki AR, Morelli MP, Diamantopoulos L, Singh N, Grivas P. Association of blood 
biomarkers and autoimmunity with immune related adverse events in patients with cancer 
treated with immune checkpoint inhibitors. Sci Rep. 2021 Apr 27;11(1):9029.  
10. Chennamadhavuni A, Abushahin L, Jin N, Presley CJ, Manne A. Risk Factors and Biomarkers for 
Immune-Related Adverse Events: A Practical Guide to Identifying High-Risk Patients and 
Rechallenging Immune Checkpoint Inhibitors. Front Immunol. 2022;13:779691.  
11. von Itzstein MS, Khan S, Gerber DE. Investigational Biomarkers for Checkpoint Inhibitor Immune-
Related Adverse Event Prediction and Diagnosis. Clin Chem. 2020 Jun;66(6):779–93.  
12. Albarel F, Castinetti F, Brue T. MANAGEMENT OF ENDOCRINE DISEASE: Immune check point 
inhibitors-induced hypophysitis. European Journal of Endocrinology. 2019 Sep 1;181(3):R107–
18.  
13. Liu R, Peng L, Zhou L, Huang Z, Zhou C, Huang C. Oxidative Stress in Cancer Immunotherapy: 
Molecular Mechanisms and Potential Applications. Antioxidants (Basel). 2022 Apr 27;11(5):853.  
14. Sangiuliano B, Pérez NM, Moreira DF, Belizário JE. Cell Death-Associated Molecular-Pattern 
Molecules: Inflammatory Signaling and Control. Mediators Inflamm. 2014;2014:821043.  
15. Adam C, Wohlfarth J, Haußmann M, Sennefelder H, Rodin A, Maler M, et al. Allergy-Inducing 
Chromium Compounds Trigger Potent Innate Immune Stimulation Via ROS-Dependent 
Inflammasome Activation. J Invest Dermatol. 2017 Feb;137(2):367–76.  
16. Peng W, Williams LJ, Xu C, Melendez B, McKenzie JA, Chen Y, et al. Anti-OX40 Antibody Directly 
Enhances The Function of Tumor-Reactive CD8+ T Cells and Synergizes with PI3Kβ Inhibition in 
PTEN Loss Melanoma. Clin Cancer Res. 2019 Nov 1;25(21):6406–16.  

 
 
 
BIO-CHECKPOINT 
 
   
 
20 
Final Version 1.2 12th September 2023-BIOCHECKPOINT 
 
17. Murphy MP, Siegel RM. Mitochondrial ROS fire up T cell activation. Immunity. 2013 Feb 
21;38(2):201–2.  
18. Li Y, Liang R, Zhang X, Wang J, Shan C, Liu S, et al. Copper Chaperone for Superoxide Dismutase 
Promotes Breast Cancer Cell Proliferation and Migration via ROS-Mediated MAPK/ERK Signaling. 
Front Pharmacol. 2019;10:356.  
19. Chamoto K, Chowdhury PS, Kumar A, Sonomura K, Matsuda F, Fagarasan S, et al. Mitochondrial 
activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent 
antitumor activity. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):E761–70.  
20. Mao D, Hu F, Yi Z, Kenry K, Xu S, Yan S, et al. AIEgen-coupled upconversion nanoparticles 
eradicate solid tumors through dual-mode ROS activation. Sci Adv. 2020 Jun 26;6(26):eabb2712.  
21. Di Dalmazi G, Hirshberg J, Lyle D, Freij JB, Caturegli P. Reactive oxygen species in organ-specific 
autoimmunity. Auto Immun Highlights. 2016 Aug 4;7(1):11.  
22. Conroy M, Naidoo J. Immune-related adverse events and the balancing act of immunotherapy. 
Nat Commun. 2022 Jan 19;13(1):392.  
23. Malonaldehyde (malondialdehyde). IARC Monogr Eval Carcinog Risks Hum. 1999;71 Pt 3(PT 
3):1037–47.  
24. Sobsey CA, Han J, Lin K, Swardfager W, Levitt A, Borchers CH. Development and evaluation of a 
liquid chromatography–mass spectrometry method for rapid, accurate quantitation of 
malondialdehyde in human plasma. Journal of Chromatography B. 2016 Sep 1;1029–1030:205–
12.  
25. Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic 
molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis. 2005 
Aug;15(4):316–28.  
26. Jansen HI, Bruinstroop E, Heijboer AC, Boelen A. Biomarkers indicating tissue thyroid hormone 
status: ready to be implemented yet? Journal of Endocrinology. 2022 May 1;253(2):R21–45.  
 
 
 

 
 
 
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17.0  APPENDICIES 
17.1 
Appendix 2 – Amendment History 
Amendment 
No. 
Protocol 
version no. 
Date issued 
Author(s) of 
changes 
Details of changes made 
1 
1.1 
10/08/2023 
L Duvall 
Amended frequency of when to send list of 
NHS numbers to MyCall 
2 
1.2 
12/09/2023 
L Duvall 
Included patient consent process 
 
List details of all protocol amendments here whenever a new version of the protocol is produced. 
Protocol amendments must be submitted to the Sponsor for approval prior to submission to the 
REC.