Prediction of Recurrent Events With 18F-Fluoride to Identify Ruptured and High-risk Coronary Artery Plaques in Patients With Myocardial Infarction
Conditions: Coronary Artery Disease, Myocardial Infarction
Sponsor: University of Edinburgh

 
 
  
Study Protocol  
 
Prediction of Recurrent Events with 18F-Fluoride to Identify Ruptured 
and High-risk Coronary Artery Plaques in Patients with Myocardial 
Infarction  
  
Co-sponsors  
University of Edinburgh & NHS Lothian  
ACCORD  
The Queen’s Medical Research Institute  
47 Little France Crescent  
Edinburgh  
EH16 4TJ  
Funder  
Wellcome Trust   
Funding Reference 
Number  
WT103782AIA - Senior Investigator Award  
Chief Investigator  
Professor David Newby  
REC Number  
15-SS-0059  
EudraCT Number  
2014-004021-41  
ClinicalTrials.gov ID 
number  
NCT02278211  
Number and Date  
Version  11.0 - 24 February 2022  
  

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COORDINATING CENTRE  
Chief Investigator  
Professor David E Newby  
Centre of Cardiovascular Science  
Chancellor’s Building  
51 Little France Crescent  
Edinburgh EH16 4SB   
Tel: 0131 242 6515   
Fax: 0131 242 6379   
Email: d.e.newby@ed.ac.uk  
Co Investigator  
Dr Marc Dweck  
Centre of Cardiovascular Science  
Chancellor’s Building  
51 Little France Crescent  
Edinburgh EH16 4SB   
  
Tel: 0131 242 6364  
Email: marc.dweck@nhslothian.scot.nhs.uk  
Clinical Research Fellow  
Dr Marwa Daghem  
Centre of Cardiovascular Science  
Chancellor’s Building  
51 Little France Crescent  
Edinburgh EH16 4SB   
  
  
Tel: 07500931090   
Email: mdaghem@exseed.ed.ac.uk  
Co-sponsor Representative  
Dr  Fiach O’Mahony  
University of Edinburgh   
Queen’s Medical Research Institute  
47 Little France Crescent  
Edinburgh EH16 4SB   
  
Tel: 0131 242  6226   
Fax: 0131 242 9418  
Email:  Fiach.O’Mahony@ed.ac.uk  
Trial Manager   
Claire Battison  
Edinburgh Clinical Trials Unit  
University of Edinburgh  
NINE, 9 Little France Road  
Edinburgh EH16 4UX  
  
Tel: 0131 651 9919  
Email: c.battison@ed.ac.uk  
  
Trial Statistician  
Professor Steff Lewis  
Edinburgh Clinical Trials Unit  
University of Edinburgh  
NINE, 9 Little France Road  
Edinburgh EH16 4UX  
  
Tel: 0131 651 9956   
Email: steff.lewis@ed.ac.uk  
  
  
 
 
 
 
 
 
 
 
 
 
 
 

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CONTENTS  
1  
INTRODUCTION ........................................................................................ 8  
 
1.1  
BACKGROUND ................................................................................. 8  
1.1.1 Pathogenesis of Coronary Artery Disease ....................................... 8 
1.1.2 Non-invasive Imaging of the Vulnerable Plaque .............................. 8 1.1.3 
Recurrent Events Following Acute Myocardial Infarction ................. 9 1.1.4 
Stratified Medicine and Intervention .............................................. 10 1.1.5 
Coronary Microcalcification Activity ............................................... 10  
1.2  RESEARCH HYPOTHESIS ............................................................. 11 1.3 
 
RATIONALE FOR THE STUDY ....................................................... 11  
2  
STUDY OBJECTIVES .............................................................................. 11  
 
2.1  
OBJECTIVES .................................................................................. 11  
2.1.1 Primary Objective .......................................................................... 11 2.1.2 
Secondary Objectives ................................................................... 11 2.1.3 
Safety Objectives .......................................................................... 12 2.1.4 
Exploratory Objectives .................................................................. 12  
 
2.2  
ENDPOINTS .................................................................................... 12  
2.2.1 Primary Endpoints ......................................................................... 12 2.2.2 
Secondary Endpoints .................................................................... 12 2.2.3 
Safety Endpoints ........................................................................... 13  
3 
STUDY DESIGN ....................................................................................... 13  
3.1 
RATIONALE FOR STUDY DESIGN ................................................. 13  
4 
STUDY POPULATION ........................................................................... 13  
4.1 
NUMBER OF PARTICIPANTS ......................................................... 13 4.2      
INCLUSION CRITERIA..................................................................... 13 4.3       
EXCLUSION CRITERIA .................................................................. 13 4.4       
CO-ENROLMENT ............................................................................ 14  
5 
PARTICIPANT SELECTION AND ENROLMENT .................................... 14  
5.1 
IDENTIFYING PARTICIPANTS ....................................................... 14 5.2 
 
CONSENTING PARTICIPANTS ...................................................... 14 
5.3  SCREENING FOR ELIGIBILITY ...................................................... 14 
5.4  INELIGIBLE AND NON-RECRUITED PARTICIPANTS .................... 
15 5.5  
PROCEDURES FOR HANDLING PARTICIPANTS 
INCORRECTLY  
ENROLLED ..................................................................................... 15  
 
5.6  
WITHDRAWAL OF STUDY PARTICIPANTS ................................... 15  
6 
INVESTIGATIONAL MEDICINAL PRODUCT .......................................... 15  
 
6.1  
STUDY DRUG ................................................................................. 15  
6.1.1 Study Drug Identification ............................................................... 15 6.1.2 
Study Drug Manufacturers ............................................................ 15 6.1.3 
Marketing Authorisation Holder ..................................................... 16  
6.1.4 Labelling and Packaging ............................................................... 16  

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6.2  DOSING REGIME ............................................................................ 17 6.3 
 
OVERDOSE .................................................................................... 17 6.4 
 
OTHER MEDICATIONS ................................................................... 17  
6.4.1 Non-Investigational Medicinal Products ......................................... 17 
6.4.2 Permitted Medications ................................................................... 17  
7  
STUDY ASSESSMENTS ......................................................................... 18  
7.1  SAFETY ASSESSMENTS ............................................................... 19 7.2 
 
STUDY ASSESSMENTS ................................................................. 20  
7.2.1 Screening Assessment ................................................................. 20 7.2.2 
Baseline Assessment .................................................................... 20 7.2.3 
Follow Up Assessments ................................................................ 20 7.2.4 
Positron Emission and Computed Tomography Coronary  
Angiography .................................................................................. 20  
 
7.3  
SUB-STUDY ASSESSMENTS ......................................................... 21  
7.3.1 Short-term Reproducibility (PRE18FFIR-REPRO) .......................... 21 7.3.2 
Time Course (PRE18FFIR-TIME) ................................................... 21 7.3.3 In 
Vivo Plaque Characterisation (PRE18FFIR-IMAGE) ................... 21  
8 
DATA COLLECTION ............................................................................... 22  
9 
DATA MANAGEMENT ............................................................................. 22  
9.1.1 Personal Data ............................................................................... 22 9.1.2 
Data Information Flow ................................................................... 22 9.1.3 
Transfer of Data ............................................................................ 23 9.1.4 Data 
Controller .............................................................................. 23 9.1.5 Data 
Breaches .............................................................................. 23  
10 
STATISTICS AND DATA ANALYSIS ...................................................... 23  
10.1  
SAMPLE SIZE CALCULATION ........................................................ 23 
10.2  
PROPOSED ANALYSES ................................................................. 23  
10.2.1 Description of Analysis Sets .......................................................... 23 
10.2.2 Methods of Statistical Analysis ...................................................... 24  
11  
ADVERSE EVENTS ................................................................................. 24  
11.1  
DEFINITIONS .................................................................................. 24 
11.2  
IDENTIFYING AEs AND SAEs ........................................................ 25 
11.3  
RECORDING AEs AND SAEs ......................................................... 25 
11.4  
ASSESSMENT OF AEs AND SAEs ................................................. 26  
11.4.1 Assessment of Seriousness .......................................................... 26 
11.4.2 Assessment of Causality ............................................................... 26 
11.4.3 Assessment of Expectedness ....................................................... 26 
11.4.4 Assessment of Severity ................................................................. 26  
 
11.5  
REPORTING OF SAEs/SARs/SUSARs ........................................... 27  
11.6  
REGULATORY REPORTING REQUIREMENTS ............................. 
27 11.7  
FOLLOW UP PROCEDURES .......................................................... 27  
12 
PREGNANCY........................................................................................... 28  
13 
TRIAL MANAGEMENT AND OVERSIGHT ARRANGEMENTS............... 28  

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13.1  
TRIAL MANAGEMENT GROUP ...................................................... 28 
13.2  
TRIAL STEERING COMMITTEE ..................................................... 28 
13.3  
DATA MONITORING COMMITTEE ................................................. 28 
13.4  
INSPECTION OF RECORDS .......................................................... 28 
13.5  
RISK ASSESSMENT ....................................................................... 28 
13.6  
BENEFIT/RISK BALANCE ............................................................... 29  
13.6.1 Benefits ......................................................................................... 29 
13.6.2 Risks ............................................................................................. 29  
 
13.7  
STUDY MONITORING AND AUDIT ................................................. 29  
14  
GOOD CLINICAL PRACTICE .................................................................. 30  
14.1  
ETHICAL CONDUCT ....................................................................... 30 
14.2  
REGULATORY COMPLIANCE ........................................................ 30 
14.3  
INVESTIGATOR RESPONSIBILITIES ............................................. 30  
14.3.1 Informed Consent ......................................................................... 30 
14.3.2 Study Site Staff ............................................................................. 31 
14.3.3 Data Recording ............................................................................. 31 
14.3.4 Investigator Documentation ........................................................... 31 
14.3.5 GCP Training ................................................................................ 31 
14.3.6 Confidentiality ............................................................................... 31 
14.3.7 Data Protection ............................................................................. 31  
15 
STUDY CONDUCT RESPONSIBILITIES ................................................. 32  
15.1 
PROTOCOL AMENDMENTS ........................................................... 32 
15.2  
PROTOCOL VIOLATIONS AND DEVIATIONS ................................ 
32 15.3  
SERIOUS BREACH REQUIREMENTS ........................................... 32 
15.4  
STUDY RECORD RETENTION ....................................................... 32 
15.5  
END OF STUDY .............................................................................. 33 
15.6  
INSURANCE AND INDEMNITY ....................................................... 33  
16 
REPORTING, PUBLICATIONS AND NOTIFICATION OF RESULTS ...... 34  
16.1 
AUTHORSHIP POLICY ................................................................... 34 
16.2  
PUBLICATION ................................................................................. 34 
16.3  
PEER REVIEW ................................................................................ 34  
17 
REFERENCES ......................................................................................... 35  
  
 
 
 
 
 
 
 
 
 
 
 
 
 

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PROTOCOL APPROVAL  
PRE18FFIR Study  
  
Signatures  
  
Professor David Newby  
  
  
  
 
Chief Investigator  
Signature  
 
Date 
 
 
 
 
 
 
 
 
 
 
 
 
Professor Steff Lewis  
 
 
 
 
Study Statistician  
Signature  
 
Date 
 
 
 
 
 
 
 
 
 
 
 
 
Dr.Fiach O’Mahony  
 
 
 
 
Sponsor(s)  
Representative  
Signature  
 
Date  
 
 
 
 
 
 
 
 
 
 
 
 
 
___________________  
 
___________ 
Principal Investigator  
Signature  
 
Date  

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LIST OF ABBREVIATIONS  
ACCORD  
Academic and Clinical Central Office for Research & 
Development - Joint office for University of Edinburgh 
and NHS Lothian  
AE  
Adverse Event  
AR  
Adverse Reaction  
CI  
Chief Investigator  
CT  
Computer Tomography  
CTA  
Clinical Trial Authorisation  
CTCA  
Computer Tomography Coronary Angiography  
CTIMP  
Clinical Trial of an Investigational Medicinal Product  
CRF  
Case Report Form  
DNA  
Deoxyribose Nucleic Acid  
eGFR  
Estimated Glomerular Filtration Rate  
GCP  
Good Clinical Practice  
ICH  
International Conference on Harmonisation  
IMP  
Investigational Medicinal Product  
ISF  
Investigator Site File  
IVUS  
Intravascular Ultrasound  
MHRA  
Medicines and Healthcare products Regulatory Authority  
NIMP  
Non Investigational Medicinal Product  
NIRS  
Near Infrared Spectroscopy  
OCT  
Optical Coherence Tomography  
PET  
Positron Emission Tomography  
R&D  
Research & Development  
REC  
Research Ethics Committee  
SAE  
Serious Adverse Event  
SAR  
Serious Adverse Reaction  
SOP  
Standard Operating Procedure  
SUSAR  
Suspected Unexpected Serious Adverse Reaction  
TMF  
Trial Master File  
UAR  
Unexpected Adverse Reaction  
  
  

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1  INTRODUCTION  
1.1 BACKGROUND  
1.1.1 Pathogenesis of Coronary Artery Disease  
Coronary plaque rupture of mildly stenotic coronary atherosclerosis is the 
commonest cause of acute coronary thrombosis, myocardial infarction and 
death [Davies, 2000]. It is closely linked to hypercholesterolaemia and has a 
dominant inflammatory phenotype. Classical histological features include a 
lipid-rich pool, thin fibrous cap, paucity of vascular smooth muscle cells and an 
intense inflammatory cell infiltrate [Davies, 2000].  
Plaque rupture does not invariably lead to thrombotic occlusion of the coronary 
artery. Coronary plaque events are very common, and the majority of such 
events do not cause coronary occlusion [Mann & Davies, 1999; Davies, 2000]. 
Here, plaque rupture and thrombosis are organised, remodelled and 
incorporated into the atherosclerotic plaque itself. Indeed, 80% of plaques that 
cause over 50% diameter stenosis have evidence of old healed plaque rupture 
with incorporation of thrombus into the atheroma [Mann & Davies, 1999]. This 
process contributes to the progression of coronary atherosclerosis and explains 
the first appearance, or step change in severity, of angina pectoris.  
Coronary plaque events are often not isolated. Plaque rupture can occur 
throughout the body and is not confined to a single vascular bed. Moreover, in 
patients with acute coronary syndromes, it is common for multiple plaque 
events to occur simultaneously and beyond the culprit lesion itself. Post-mortem 
studies indicate that, on average, 2.4 coronary thrombotic events occur in each 
patient who presents with fatal coronary heart disease [Mann & Davies, 1999; 
Davies, 2000]. This suggests that systemic and generalised mechanisms and 
mediators play an important role in addition to local factors within the coronary 
artery wall.  
In summary, coronary atherosclerosis is characterised by multiple and recurrent 
plaque rupture events that are often sub-clinical and cause step wise growth of 
the plaque as it heals and remodels. Patients undergoing repeated plaque 
rupture events are likely to be at increased risk of myocardial infarction.  
1.1.2 Non-invasive Imaging of the Vulnerable Plaque  
In the study of coronary artery disease, many researchers have searched for a 
non-invasive imaging biomarker of plaque vulnerability and rupture. For the first 
time, we have demonstrated that 18F-fluoride positron emission tomography 
can detect high-risk coronary plaque in patients with stable coronary artery 
disease [Joshi et al, 2014].   
In the vasculature, 18F-fluoride acts as a marker of novel calcification activity 
[Dweck et al, 2012a; Dweck et al, 2013]. Similar to other conditions, calcification 
in coronary atheroma occurs as a healing response to intense necrotic 
inflammation, making 18F-fluoride a useful marker of high-risk atherosclerotic 
plaque. We have previously demonstrated increased uptake of this tracer in the 
coronary vasculature localizing to individual coronary lesions and identifying 
patients with increased cardiovascular risk factor profiles [Dweck et al, 2012b]. 

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More recently we have conducted a prospective study of 40 patients with 
myocardial infarction in whom 18F-fluoride localised to the culprit plaque 
(Figure 1) in over 90% of patients [Joshi et al, 2014]. This finding was confirmed 
in 12 patients with a recent stroke undergoing carotid endartectomy where 
18Ffluoride uptake was observed at the site of plaque rupture in 100% of 
patients and this uptake correlated with increased calcification activity and 
areas of necrosis on histology. Finally, we studied 40 patients with stable 
coronary artery disease. Increased uptake was observed in 45% of these 
patients and this again localised to individual coronary plaques (Figure 2). 
Interestingly these lesions were associated with multiple high-risk markers on 
radiofrequency and gray-scale intravascular ultrasound (necrotic core, positive 
remodeling and microcalcification). Importantly, plasma high-sensitivity 
troponin concentrations were much higher in patients with 18F-fluoride positive 
plaques compared to patients without evidence of uptake (7.89±9.34 versus 
3.10±1.89 ng/L, P=0·047). The latter observation is of particular interest as it 
supports the hypothesis that 18F-fluoride is detecting subclinical plaque rupture 
in those with stable disease, similar to its mechanism of activity following 
myocardial infarction. Moreover, plasma troponin concentrations measured by 
a highsensitivity assay also predict an adverse outcome amongst patients with 
stable coronary artery disease [Omland et al, 2013] and provide a useful 
surrogate biomarker of therapeutic efficacy.  
1.1.3 Recurrent Events Following Acute Myocardial Infarction  
Myocardial infarction and stroke are two of the commonest causes of 
hospitalisation, major disability and death in the United Kingdom. However, 
despite many billions of pounds being spent each year on their treatment, 
recurrent cardiovascular events are common. Indeed, within one year of acute 
myocardial infarction, one in five patients have recurrent events despite optimal 
medical therapy [Mills et al, 2011], and one half of these recurrent events occur 
at sites not felt to have caused their initial presentation [Stone et al, 2011]. 
Coronary calcification is pathognomonic of atherosclerosis and recent studies 
in Egyptian mummies have demonstrated that it has been present in humans 
for millennia [Thompson et al, 2013]. Despite this, the mechanisms of its 
formation have yet to be precisely defined. Furthermore, the nature and extent 
of vascular calcification may vary according to the pathophysiological setting 
and underlying disease process. Given that it is independently associated with 
a 3 to 4-fold increased risk of death [Detrano et al, 2008], a better understanding 
of the pathophysiology and mechanisms of vascular calcification will be critical 
to our understanding of cardiovascular disease. This is particularly important if 
we are to determine its role in the initiation, progression and consequences of 
atherosclerosis. This may provide novel insights into the pathophysiology of 
atherosclerosis as well as identify new and as yet unexplored pathways that 
could be the target for future life-saving therapeutic interventions. For example, 
although highly successful in treating cardiovascular disease, statins only 
prevent 25% of recurrent cardiovascular events, and have no effect on the 
progression of vascular calcification. The majority of recurrent events therefore 

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remain unchecked and we urgently need more effective and comprehensive 
interventions.  
Non-invasive imaging techniques to detect early active vascular calcification in 
atherosclerosis have the potential to identify high-risk and ruptured 
atherosclerotic plaques, thereby providing a major novel biomarker of plaque 
vulnerability. This will provide a platform to explore and follow the in vivo 
pathogenesis of atherosclerosis, as well as assess novel anti-atherosclerotic 
interventions. This will also facilitate the more accurate identification of culprit 
atherosclerotic plaque in the precipitation of myocardial infarction and stroke, 
allowing better delivery and targeting of our therapies at both the lesion and 
patient level. This approach would potentially be a major advance for the 
treatment of unstable atherosclerotic disease.  
1.1.4 Stratified Medicine and Intervention  
Current treatment decisions for patients with acute myocardial infarction are 
based on a large number of major multi-centre international randomised 
controlled trials. These trials demonstrate major secondary preventative 
benefits with the use of anti-platelet therapy, statins, angiotensin-converting 
enzyme inhibition and beta-blockade following acute myocardial infarction. 
However, all patients are treated with the same combination of medications with 
little attempt to risk stratify patients and select those who have the most to 
benefit from such therapies.   
Emergency or immediate coronary angiography with a view to coronary 
revascularisation is now recommended in the vast majority of patients 
presenting with acute myocardial infarction. Indeed, 80% of patients admitted 
with non-ST segment elevation myocardial infarction in the United Kingdom 
undergo coronary angiography with a view to coronary revascularisation during 
their 
index 
hospital 
admission 
[MINAP, 
2013]. 
However, 
coronary 
revascularisation following myocardial infarction focuses on treating flowlimiting 
stenoses (≥70% luminal stenosis) with coronary revascularisation strategies 
such as percutaneous coronary intervention and coronary artery bypass graft 
surgery. However, we know that the majority of plaque rupture events occur on 
non-flow limiting stenoses (see section 1.1), and this observation questions 
whether we are over treating a large number of patients and indeed coronary 
artery lesions without actually tackling the future potential culprit lesions. Using 
18F-fluoride, we have “the opportunity to better assess the commonly accepted 
belief that most myocardial infarctions are caused by rupture of previously non-
obstructive plaques” [Thomas & Haraszti, 2014].  
1.1.5 Coronary Microcalcification Activity  
In preliminary proof-of-concept studies, we have assessed a global measure of 
coronary 18F-fluoride uptake in patients with coronary artery disease. We have 
termed this measure ‘coronary microcalcification activity’ and is analogous to 
the computed tomography-derived Agatston coronary artery calcium score. We 
have shown that total coronary microcalcification activity can be measured in a 
reproducible manner [Tzolos et al, 2020], and that it correlates with measures 
of high-risk low-attenuation plaque [Kwiecinski et al, 2020a]. In a post-hoc 

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analysis of nearly 300 patients with coronary artery disease from 2 centres, 
coronary microcalcification activity was the strongest predictor of fatal or 
nonfatal myocardial infarction over 42 months of follow up [Kwiecinski et al, 
2020b]. It outperformed all standard cardiovascular risk factors, risk scores and 
standard measures of coronary artery disease severity.  
1.2 RESEARCH HYPOTHESIS  
The central hypothesis of the PRE18FFIR study is that coronary 18F-fluoride 
uptake can identify high-risk and ruptured atherosclerotic plaque in patients with 
recent myocardial infarction. This has the potential to assist in the diagnosis, 
risk stratification, investigation, management and treatment of patients with 
acute myocardial infarction.  
1.3 RATIONALE FOR THE STUDY   
We have investigated and described the use of 18F-fluoride positron emission 
tomography and computed tomography coronary angiography to identify 
highrisk or ruptured coronary atherosclerotic plaque [Joshi et al. Lancet 2014]. 
This is the first non-invasive imaging technique to achieve this and it has been 
heralded as a major advance with wide reaching ramifications for the diagnosis, 
investigation, and treatment of coronary artery disease [Thomas & Haraszti, 
2014]. Importantly, it may provide the basis of a new paradigm in the treatment 
and management of patients with myocardial infarction by tailoring therapy to 
the presence and extent of plaque inflammation and rupture rather than the 
anatomical severity of luminal stenoses. More targeted and focused 
investigations and interventions have the potential to shorten hospital stay, to 
avoid the indiscriminate application of expensive therapies, and to focus and to 
maximise the impact of our current successful therapies to reduce recurrent 
major adverse cardiac events.  
The study rationale is therefore to confirm our preliminary findings in a broad 
range of patients across different centres throughout the United Kingdom and 
across the world. Specifically, the study is intended to confirm whether coronary 
18F-fluoride uptake identifies high-risk or ruptured coronary atherosclerotic 
plaque, and to determine if coronary 18F-fluoride uptake is predictive of disease 
progression and clinical outcome.  We have also planned two sub-studies to 
assess the reproducibility and natural history of 18F-fluoride uptake and an 
additional sub-study to provide correlation with in vivo plaque characterisation 
tests.  These sub-studies are described in more detail in separate protocols.   
2  STUDY OBJECTIVES  
2.1 OBJECTIVES  
2.1.1 Primary Objective  
 To determine whether coronary 18F-fluoride uptake is associated with 
major adverse cardiac events in patients with multi-vessel coronary 
artery disease and recent myocardial infarction.  

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2.1.2 Secondary Objectives  
In patients with multi-vessel coronary artery disease and recent myocardial 
infarction, to determine whether coronary 18F-fluoride uptake:  
• 
Is associated with secondary clinical outcomes including all-cause 
death, cardiac death, non-fatal recurrent myocardial infarction, and, 
unscheduled coronary revascularisation..  
• 
Can identify areas of high-risk or ruptured atherosclerotic plaque in a 
large broad population of patients across multiple sites.  
• 
Can predict the territory of subsequent myocardial reinfarction.  
• 
Can identify areas of future progression of coronary atherosclerotic 
plaque (volume and calcification)  
2.1.3 Safety Objectives  
• 
To determine the radiation exposure and effective dose associated with 
18F-fluoride positron emission tomography and computed tomography 
coronary angiography.  
• 
To identify adverse events associated with 18F-fluoride positron 
emission tomography and computed tomography coronary angiography.  
2.1.4 Exploratory Objectives  
We will initially explore whether the identification of 18F-fluoride positive 
coronary atherosclerotic plaque has the potential to guide patient management. 
Here positron emission tomography and computed tomography coronary 
angiography results will be compared with the subsequent application of 
coronary revascularisation and optimal medical therapy. Importantly, we will 
also explore the feasibility of using the identification of 18F-fluoride positive 
coronary atherosclerotic plaque to guide coronary revascularisation and 
intervention. For example, how many stenotic but 18F-fluoride negative lesions 
undergo intracoronary stenting, and how many 18F-fluoride positive lesions are 
left untreated and are responsible for recurrent cardiovascular events?   
  
2.2 ENDPOINTS  
2.2.1 Primary Endpoints  
 Cardiac death, non-fatal recurrent myocardial infarction, or unscheduled 
coronary revascularisation.  
2.2.2 Secondary Endpoints  
These will include:  
• 
All-cause death  
• 
Cardiac death  
• 
Non-fatal recurrent myocardial infarction  
• 
Unscheduled coronary revascularisation  

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• 
18F-NaF uptake localisation to culprit plaque causing the index 
myocardial infarction  
• 
Territory of subsequent myocardial reinfarction.  
• 
Coronary artery plaque progression by computed tomography coronary 
angiography. Plaque volume, composition and calcification.  
  
2.2.3 Safety Endpoints  
  
• 
Dose-length product and effective radiation dose associated with PET 
and CT scans.  
• 
Adverse events experienced during the scan or self-reported within 48 
hours of PET and CT scans.  
3  STUDY DESIGN  
This is a multicentre study of an investigational medical product with longitudinal 
follow-up for disease progression and clinical outcomes.  
3.1  
RATIONALE FOR STUDY DESIGN  
We wish to confirm our preliminary findings in a larger multicentre study of 
patients with recent myocardial infarction. We will undertake a study in patients 
with recent myocardial infarction who will undergo coronary 18F-fluoride 
positron 
emission 
tomography 
and 
computed 
tomography 
coronary 
angiography immediately following hospitalisation. We will conduct a 
longitudinal cohort follow-up of participants who will undergo clinical follow-up 
of at least 2 years duration when repeat computed tomography coronary 
angiography will be performed.   
  
Before conducting a randomised controlled trial using coronary 18F-fluoride 
uptake to stratify and guide treatment, it is imperative that we confirm the 
diagnostic and prognostic importance of coronary 18F-fluoride uptake within a 
large multicentre study.  
4   STUDY POPULATION  
4.1      NUMBER OF PARTICIPANTS  
We will recruit approximately 700 patients with recent myocardial infarction and 
multi-vessel coronary heart disease.4.2      INCLUSION CRITERIA  
For inclusion in the study subjects will fulfil the following criteria:  
1. 
Patients aged ≥50 years with recent (<21 days) type 1 myocardial 
infarction and angiographically proven multi-vessel coronary artery 
disease defined as at least two major epicardial vessels with any 
combination of either (a) >50% luminal stenosis, or (b) previous 

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revascularisation (percutaneous coronary intervention or coronary 
artery bypass graft surgery).  
2. 
Provision of informed consent prior to any study specific procedures  
4.3       EXCLUSION CRITERIA  
Subjects will not enter the study if any of the following exclusion criteria are 
fulfilled:  
  
1. 
Inability or unwilling to give informed consent  
2. 
Women who are pregnant, breastfeeding or of child-bearing potential 
(women who have experienced menarche, are pre-menopausal and  
have not been sterilised) will not be enrolled into the trial  
3. 
Major intercurrent illness with life expectancy <2 year  
4. 
Renal dysfunction (estimated glomerular filtration rate ≤30 mL/min/1.73 
m2)  
5. 
Contraindication to iodinated contrast agent, positron emission 
tomography or computed tomography  
6. 
Permanent or persistent atrial fibrillation  
7. 
Previous screening as part of the trial   
4.4       CO-ENROLMENT  
Co-enrolment with a non-CTIMP or another CTIMP clinical trial may be allowed 
provided this is not expected to place an undue burden upon participants and 
their families and will not compromise the primary end point of either trial.  
Consideration will also be given to the total exposure to ionising radiation should 
additional studies require further exposure.  
Co-enrolment with another CTIMP will only be permitted with agreement of the 
Sponsors, Trial Steering Committees, and Chief Investigators of both studies.  
5   PARTICIPANT SELECTION AND ENROLMENT  
5.1 IDENTIFYING PARTICIPANTS  
Patients presenting with myocardial infarction will be approached for study 
recruitment by their usual care team. This will normally be undertaken during 
their index hospital admission. Patients will be provided with a Patient 
Information Sheet and given an opportunity to ask questions about participation 
in the trial.   
5.2 CONSENTING PARTICIPANTS  
After an appropriate length of time, patients willing to participate in the study will 
be asked to consent and undergo screening. Written informed consent will be 

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obtained by a suitably qualified member of the research team before any study 
related procedures are performed.   
5.3 SCREENING FOR ELIGIBILITY  
Screening for eligibility based on pre-existing clinical investigations (i.e. 
standard clinical biochemical and haematological investigations and coronary 
angiographic findings) will be performed by a member of the research team and 
will only take place once written consent has been obtained. Where it is not 
possible for participants to be consented during their initial hospital admission, 
they may be asked to attend an additional study visit during which consent will 
be taken and eligibility confirmed. Once a patient has agreed to participate and 
is deemed eligible, they will be invited to attend the baseline visit for 18Ffluoride 
positron emission tomography and computed tomography coronary angiogram 
scans.  
5.4 INELIGIBLE AND NON-RECRUITED PARTICIPANTS  
Ineligible and non-recruited patients will receive standard medical care. An 
anonymised log will be kept of patients who were screened for the study and 
subsequently found to be ineligible or not recruited.    
5.5 PROCEDURES FOR HANDLING PARTICIPANTS INCORRECTLY  
ENROLLED  
Patients identified as being incorrectly enrolled prior to their initial CT-PET scan 
will be excluded from the primary study analysis and replaced. Patients who are 
identified as incorrectly enrolled but have undergone their initial CT-PET will be 
included in the primary analysis and we will endeavour to continue to collect 
study data without involving the participant in any further study related 
procedures.  
5.6 WITHDRAWAL OF STUDY PARTICIPANTS  
Participants are free to withdraw from the study at any point or a participant can 
be withdrawn by the investigator. If withdrawal occurs, the primary reason for 
withdrawal will be documented in the participant’s case record form, if possible. 
The patient will have the option of withdrawal from (i) all or specific further study 
procedures but continued collection of routine clinical and safety data, (ii) all 
further aspects of the study with no further collection of any data, and (iii) all 
aspects of the trial with removal of all previously collected data.    
 

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6  INVESTIGATIONAL MEDICINAL PRODUCT   
6.1 STUDY DRUG  
Investigational 
product  
Dosage form and 
strength  
Manufacturer  
[18F] Sodium Fluoride  
250 MBq  
See 6.1.2  
6.1.1 Study Drug Identification  
[18F] Sodium Fluoride (18F-NaF) sterile solution for injection  
  
6.1.2 Study Drug Manufacturers  
Manufacturing will be performed by:  
  
  
Clinical Research Imaging Centre (CRIC)  
  
University of Edinburgh  
  
47 Little France Crescent   
  
Edinburgh  
  
EH16 4TJ  
  
Licence: MIA (IMP) 1384   
  
PETNET Solutions  
Lesley Harrison Building  
Mount Vernon Hospital  
Northwood  
Middlesex  
HA6 2RN  
Licence: MIA (IMP) 21750  
  
PETNET Solutions  
Nottingham City Hospital  
Gate 1  
Hucknall Road  
Nottingham  
NG5 1PB  
Licence: MIA (IMP) 21750   
6.1.3 Marketing Authorisation Holder  
There is currently no marketing authorisation for [18F] Sodium Fluoride (18F- 
NaF)  
6.1.4 Labelling and Packaging  
Labelling and packaging of the [18F] Sodium Fluoride (18F-NaF) will be carried 
out by:  
  
  
Clinical Research Imaging Centre (CRIC)  

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University of Edinburgh  
  
47 Little France Crescent   
  
Edinburgh  
  
EH16 4TJ  
  
Licence: MIA (IMP) 1384   
  
PETNET Solutions  
Lesley Harrison Building  
Mount Vernon Hospital  
Northwood  
Middlesex  
HA6 2RN  
Licence: MIA (IMP) 21750  
  
PETNET Solutions  
Nottingham City Hospital  
Gate 1  
Hucknall Road  
Nottingham  
NG5 1PB  
Licence: MIA (IMP) 21750  
  
6.2 DOSING REGIME  
Single intravenous injection  
6.3 OVERDOSE  
Given that the 18F-fluoride will be administered as a single intravenous injection 
by trained personnel, accidental or unintentional overdose is not anticipated to 
occur. Moreover, the doses and quantities of sodium fluoride are very low (<430 
µg) and >1,000-fold below the dose (3-5 mg/kg) where toxicity symptoms may 
appear and >1,000,000-fold below the anticipated lethal dose (5-10 g). 
However, if an overdose of sodium fluoride were to occur then radiation 
exposure and sodium fluoride toxicity will be the primary considerations.  
Excretion should be accelerated in order to reduce the dose taken by the 
patient.  For this reason, forced diuresis (furosemide and fluid) should be 
applied to the patient.  
  
For radiation overexposure, the patient will be contained within a safe 
environment to allow radioactive decay to occur to acceptable levels. Monitoring 
for the consequences of excess radiation exposure (such as bone marrow 
toxicity) will be undertaken as required. Symptoms of sodium fluoride toxicity 
include abdominal pain, abnormal taste (salty or soapy taste), convulsions, 
diarrhoea, drooling, headache, arrhythmia, nausea, hypopnea, bradycardia, 
tremors, vomiting, and weakness. For an intravenous overdose, there are no 
specific antidotes and supportive measures will be undertaken as clinically 

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indicated, such as hydration and anti-emetic therapy. Consideration will be 
given to intravenous calcium supplementation to compete with the excess 
fluoride ions especially in the presence of problematic arrhythmias.  
6.4 OTHER MEDICATIONS  
6.4.1 Non-Investigational Medicinal Products  
At the time of positron emission tomography and computed tomography 
coronary angiography, patients may receive oral and/or intravenous 
betablockade, such as metoprolol 5-100 mg, to slow the heart rate to below 65 
beats per minute to maximise image quality and reduce radiation exposure. 
Glyceryl trinitrate spray or tablet will be administered sublingually (200-400 µg) 
to induce coronary vasodilatation to enhance image quality of the coronary 
angiogram.  
6.4.2 Permitted Medications  
As per the standard of care for treatment of myocardial infarction, unless 
contraindicated, all patients will be encouraged to be maintained on aspirin 75 
mg once daily and maximally tolerated doses of statin, angiotensin-converting 
enzyme inhibition and beta-blocker therapy as clinically indicated and in 
accordance with local guidelines. Patients will be encouraged to be maintained 
on a P2Y12 receptor antagonist for at least 1 year.  
  
There are no study specific prohibited medications.  
7  STUDY ASSESSMENTS  
Trial participants will undergo 3 or 4 study visits (Table 1). During their initial 
hospital admission, medical notes will be screened prior to consent and 
potentially eligible patients will be approached to take part in the study. Those 
patients who are potentially eligible and wish to participate will be consented 
and routine clinical data will be collected following consent (screening visit). 
Where it is not possible for potential participants to be consented during their 
initial hospital admission, they may be asked to attend an additional study visit 
during which consent will be taken and eligibility confirmed. The participants will 
then attend the baseline visit where they will have a 18F-fluoride positron 
emission tomography, computed tomography (CT) calcium scan and CT 
coronary angiography (CTCA) scan performed before attending for a final 
clinical review and 2-year computed tomography calcium scan and CT coronary 
angiogram (2-year visit) when further routine clinical data will be collected. In 
order to assess for clinical cardiac events participants will contacted by 
telephone 1 year after their baseline PET and CTCA scans were performed.  
Following the 2-year visit, data on study outcomes will continue to be collected 
until study completion (last patient, last visit) via periodic review of the electronic 
health record and additional annual telephone review as required.  This will 
ensure that late study outcomes can be evaluated over the duration of the study.  

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In order to determine long-term outcomes, we may continue to monitor clinical 
events via annual review of patient electronic clinical records for a period of 5 
years following the completion of the participant’s final study visit.  
  
  
Table 1: Study Assessments  
  
  
Screening Visit 
1 to 30 days 
prior to 
baseline visit 
(During either 
initial hospital 
admission or  
additional study  
visit)  
Baseline  
Visit  
(Day 0)*  
Additional Visits for Reproducibility 
and Natural History Sub studies  
only  
1 Year  
Review  
(-2 weeks 
to +26 
weeks)  
2-Year 
Visit**  
 ( 0 to +  
52 
weeks)  
  
  
  
Week  
2 (±1 
week)  
Week  
12 (±2 
weeks)  
Week  
26 (±2 
weeks)  
Week  
52 (±2 
weeks)  
  
  
Eligibility 
Criteria 
and PIS  
X  
  
  
  
  
  
  
  
Consent  
X  
  
  
  
  
  
  
  
Concomitant 
medications  
X  
  
  
  
  
  
  
  
Clinical  
Assessment  
X  
  
  
  
  
  
  
  
Haematology, 
biochemistry and 
Cardiac Enzymes  
X  
This data will be collected from the existing 
patient record and should not require  
additional procedures  
  
  
ECG  
X  
  
  
GRACE Score  
Calculation  
X  
  
  
AE/SAE Reporting  
  
X  
  
  
  
  
  
X  
18F-Fluoride PET  
  
X  
X  
X  
X  
X  
X  
X  
X  
X  
  
  
CT attenuation 
correction  
  
X  
  
  
CT Calcium Scan  
  
X  
  
  
  
  
  
X  
CT Coronary 
Angiogram  
  
X  
X  
X  
X  
X  
  
X  
  
  
  
Additional PET and CT scans will 
be performed in those patients 
involved in the sub studies only  
  
  
Telephone Review  
  
  
  
  
  
  
X  
  
  
  
Additional annual telephone follow up with review of electronic 
health records if warranted until study completion (last patient, last  
visit)  
*If it is not possible for the baseline PET-CT scan to be performed within 30 days of the 
screening visit, due to either the scanner becoming unavailable or a problem with the 18F-NaF 
manufacture, then the scan will be performed within 90 days of the screening visit.  

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**Where it is not possible for the CTCA scan at the 2 year visit to be performed at the same 
time as the other 2 year visit assessments, the CTCA scan may be performed on a separate 
date.  
7.1 SAFETY ASSESSMENTS  
The dose-length product and effective radiation dose will be recorded for all 
study scans: baseline 18F-fluoride positron emission tomography and 
computed tomography coronary angiography scan, and the final 2-year 
computed tomography coronary angiogram.  
7.2 STUDY ASSESSMENTS  
This is a prospective multi-centre observational cohort study. Subjects will 
attend for clinical assessments at the screening visit (review of clinical records 
and confirmation of eligibility), baseline visit (18F-fluoride positron emission 
tomography, computed tomography calcium scan and CT coronary 
angiography scan), and at 2 years (repeat computed tomography calcium scan 
and CT coronary angiogram). Clinical outcome data will be obtained from 
routinely collected clinical data in the electronic health record and, where 
appropriate, annual telephone calls to the patients or General Practitioners.  
7.2.1 Screening Assessment  
Screening visit assessments will include clinical history, review of patient 
records to confirm study eligibility and record clinical profile, standard clinical 
biochemical and haematological variables, 12-lead electrocardiogram, the 
Global Registry of Acute Coronary Events (GRACE) score and the invasive 
coronary angiogram findings and percutaneous coronary interventional 
procedures.   
7.2.2 Baseline Assessment  
Baseline visit assessments will include 18F-fluoride positron emission 
tomography, computed tomography calcium scan and CT coronary 
angiography scan. AE/SAE’s will also be recorded.  
7.2.3 Follow Up Assessments  
Participants will be contacted by telephone at 12 months and periodically 
thereafter until the last recruited patient has completed their 2-year follow-up 
visit. The purpose of the telephone interview is to review any clinical 
cardiovascular events that may have occurred in the intervening period.  They 
will attend for a final study visit 2 years after their initial CT-PET scans. At the 
final study visit, patients will undergo a repeat computed tomography calcium 
scan and CT coronary angiogram. On-going periodic review of the electronic 
health records will continue throughout the study and may continue for a period 
of 5 years after study completion in order to assess long-term outcomes. 
Additional funding and approval will be sought if this is to continue after study 
completion.     
 
 

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7.2.4 Positron Emission and Computed Tomography  
Coronary Angiography  
Patients may receive oral and/or intravenous beta-blockade, such as metoprolol 
5-100 mg, to slow the heart rate to below 65 beats per minute to maximise 
image quality and reduce radiation exposure. Glyceryl trintrate spray or tablet 
will be administered sublingually (200-400 µg) to induce coronary vasodilatation 
to enhance image quality of the coronary angiogram.  
All patients will undergo dual cardiac and respiratory-gated positron emission 
and computed tomography imaging of the coronary arteries. Study subjects will 
be administered a target dose of 250 MBq 18F-fluoride intravenously and 
subsequently rested in a quiet environment for 60 min. An attenuation 
correction computed tomography scan will then be performed, followed by 
positron emission tomography imaging of the thorax in list-mode for 30 min.  
Computed tomography coronary calcium scan and angiography will be 
undertaken in the same visit as the 18F-fluoride scan and again at 2 years. An 
electrocardiogram-gated breath-hold computed tomography scan of the 
coronary arteries will be performed. A bolus of contrast (BMI adjusted) will be 
injected intravenously after determining the appropriate trigger delay with a test 
bolus.  Because the use of contrast is required for the CT scan study 
participants may require pre-procedural check of estimated glomerular filtration 
rate (eGFR) according to local study site imaging protocols. If, however, a 
participant develops a contraindication to the contrast agent prior to the 2-year 
computed tomography calcium scan, the computed tomography calcium scan 
can be performed without contrast.  
  
The positron emission tomography scans will be reconstructed in multiple 
phases of the cardiac cycle, with the diastolic phase (50-75%) used for analysis. 
Additional reconstructions will be undertaken as necessary. Positron emission 
tomography scans will correct for cardiac motion correction using 
electrocardiogram-gated images.  
  
Further technical details of the specific CT-PET scanning procedures and how 
inter-site imaging standardisation will be achieved will be described in a 
separate scanning document.   
  
7.3 SUB-STUDY ASSESSMENTS  
There will be three main sub-studies in relation to coronary 18F-fluoride uptake 
that will assess (i) short-term reproducibility, (ii) time course, and (iii) in vivo 
plaque characterisation. These will be performed only at the lead centre in 
Edinburgh and are described in more detail in separate protocols. Any 
participant will only be involved in one sub-study that requires a repeat 
18Ffluoride positron emission tomography and computed tomography coronary 
angiogram in addition to the main PRE18FFIR study protocol.  

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7.3.1 Short-term Reproducibility (PRE18FFIR-REPRO)  
Following the baseline assessments, a repeat 18F-fluoride positron emission 
tomography and computed tomography coronary angiogram will be performed 
within 21 days of the initial scan. This will allow the assessment of scan-rescan 
reproducibility. This will be performed in 20 patients.  
7.3.2 Time Course (PRE18FFIR-TIME)  
Following the baseline assessments, a repeat 18F-fluoride positron emission 
tomography and computed tomography coronary angiogram will be performed 
within, 12±2, 26±2 and 52±2 weeks of the initial scan. This will allow a 
description of the natural history of 18F-fluoride uptake and positivity. This will 
be performed in three cohorts of 20 patients each. Any single patient will only 
undergo a maximum of two 18F-fluoride positron emission tomography and 
three computed tomography coronary angiography scans.   
7.3.3 In Vivo Plaque Characterisation (PRE18FFIR-IMAGE)  
In 80 patients, intravascular ultrasound, near infra-red spectroscopy and optical 
coherence tomography will be performed on the culprit and control non-culprit 
lesions at the time of invasive coronary angiography. This will permit detailed 
characterisation of plaque composition and structure with which to compare the 
18F-fluoride positron emission tomography and computed tomography 
coronary angiography scans.  
8  DATA COLLECTION  
All trial data will be recorded onto paper case record forms (CRF) by a member 
of the research team and then entered into an electronic database designed 
and developed by the Edinburgh Clinical Trials Unit.   
9  DATA MANAGEMENT  
9.1.1 Personal Data  
  
The following personal data will be collected as part of the research:  
• 
Participant’s name  
• 
Address and contact details  
• 
Date of birth  
• 
GP contact details  
• 
Community Health Index (CHI) number or National Health Service (NHS) 
number.   
  

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9.1.2 Data Information Flow  
The participant’s name, address and contact details will be used by the research 
teams at the sites to contact their patients to arrange study visits. The GP 
contact details will be used by the research staff at sites to send a letter to the 
participant’s GP advising them their patient is taking part in the trial. The 
participant’s date of birth and CHI/NHS number will be collected and used to 
perform record linkage with national health registries to assess the long-term 
outcomes of heart artery inflammation. All personal data will be stored securely 
for a minimum of 5 years after the study has finished.  
  
  
9.1.3 Transfer of Data  
Data collected or generated by the study may be transferred to external 
individuals or organisations outside of the Sponsoring organisation(s).  It may 
be provided to researchers running other research studies outwith NHS 
Lothian/University of Edinburgh. Following publication of the primary paper, a 
de-identified individual participant data set will be submitted to a data archive 
for sharing purposes.  Participant information will only be used by organisations 
and researchers to conduct research in accordance with the UK Policy 
Framework for Health and Social Care Research. Where this information 
includes identifiable information, it will be held securely with strict arrangements 
about who can access the information.  
In order to perform data linkage participant’s personal details including their 
CHI number or NHS number will be transferred to national health registries, 
central NHS bodies and NHS boards.   
9.1.4 Data Controller  
A data controller is an organisation that determines the purposes for which, and 
the manner in which, any personal data are processed.  
The University of Edinburgh and NHS Lothian are joint data controllers along 
with any other entities involved in delivering the study that may be a data 
controller in accordance with applicable laws (e.g. the site)   
9.1.5 Data Breaches  
Any data breaches will be reported to the University of Edinburgh and NHS 
Lothian Data Protection Officers who will onward report to the relevant authority 
according to the appropriate timelines if required.  
10 STATISTICS AND DATA ANALYSIS  
10.1 SAMPLE SIZE CALCULATION  
Based on the assumption that one third of patients will have multiple 18Ffluoride 
positive plaques, and that the proportion of patients who have died or had a 
recurrent myocardial infarction by one year will be 30% in patients with multiple 
18F-fluoride positive plaques, and 20% in other patients, we need 692 
(231+461) subjects (power=80%, p= 2 sided 0.05). This proportion of events is 

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conservative, as we observed 20% deaths or recurrent myocardial infarctions 
at 1-year in consecutive unselected patients hospitalised with myocardial 
infarction [Mills et al, 2011; Mills et al, 2012]. In our preliminary data [Joshi et 
al, 2014], 25-45% of patients had untreated 18F-fluoride positive plaques.  This 
calculation is from a continuity corrected chi-squared test.  As the analysis will 
be time-to-event, we would expect to need 10% fewer patients, and therefore 
this calculation allows for 10% missing data.  
10.2 PROPOSED ANALYSES  
10.2.1 Description of Analysis Sets  
The primary analyses will be based on all patients who have undergone the 
initial CT-PET scan.   
10.2.2 Methods of Statistical Analysis  
The primary analysis will be performed by calculating Kaplan-Meier estimates 
of the ‘survival’ curves for the time to first event (cardiac death, recurrent 
myocardial infarction, or unscheduled coronary revascularisation) during follow 
up, and comparing these estimates for patients with and without coronary 
microcalcification activity using a log-rank test. The results will be expressed as 
the estimated hazard ratio with the corresponding 95% confidence interval and 
p value. Patients who die during follow-up whose deaths are not classified as 
cardiac deaths will be censored in the above analysis at the time of death.   
  
Since 18F-fluoride uptake is a continuous measure, a potentially more sensitive 
analysis will explore the relationship between uptake and the risk of suffering 
an event (cardiac death, recurrent myocardial infarction, or unscheduled 
coronary revascularisation). This will be performed using the Cox proportional 
hazards regression model, with (transformed) 18F-fluoride uptake as the only 
covariate. A range of potential transformations of the 18F-fluoride update will 
be explored, including fractional polynomials, to find an optimal fit to the data. 
Once an appropriate transformation for the 18F-fluoride uptake has been 
identified (as above), the Global Registry of Acute Coronary Events (GRACE) 
score will be added into the Cox proportional hazards model, to assess the 
incremental prognostic value of 18F-fluoride over and above conventional 
clinical risk markers.  
11 ADVERSE EVENTS  
The Investigator is responsible for the detection and documentation of events 
meeting the criteria and definitions detailed below.    
Full details of contraindications and side effects that have been reported 
following administration of the IMP are described in the study Investigator’s 
Brochure.  
Participants will be instructed to contact their Investigator at any time after 
consenting to join the trial if any symptoms develop.  All adverse events (AE) 

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as defined in section 11.2 that occur after joining the trial must be reported in 
detail in the Case Report Form (CRF) or AE form.  In the case of an AE, the 
Investigator should initiate the appropriate treatment according to their medical 
judgment.    
11.1 DEFINITIONS  
An adverse event (AE) is any untoward medical occurrence in a clinical trial 
participant which does not necessarily have a causal relationship with an 
investigational medicinal product (IMP).  
An adverse reaction (AR) is any untoward and unintended response to an IMP 
which is related to any dose administered to that participant.   
A serious adverse event (SAE), serious adverse reaction (SAR). Any AE or 
AR that at any dose:  
• 
results in death of the clinical trial participant  
• 
is life threatening*  
• 
requires  
in-patient  
hospitalisation**  
or  
prolongation  of 
 existing  
hospitalisation  
• 
results in persistent or significant disability or incapacity  
• 
consists of a congenital anomaly or birth defect  
• 
results in any other significant medical event not meeting the criteria 
above  
  
*Life-threatening in the definition of an SAE or SAR refers to an event where 
the participant was at risk of death at the time of the event. It does not refer to 
an event which hypothetically might have caused death if it were more severe.  
  
**Any hospitalisation that was planned prior to randomisation will not meet SAE 
criteria. Any hospitalisation that is planned post randomisation will meet the 
SAE criteria.  
  
A suspected unexpected serious adverse reaction (SUSAR) is any AR that 
is classified as serious and is suspected to be caused by the IMP, that it is not 
consistent with the information about the IMP in the Investigator’s Brochure.  
11.2 IDENTIFYING AEs AND SAEs  
There will be a 48 hour time window to collect and record all AEs and SAEs 
which occur as a result of the 18F-fluoride intravenous injection and which occur 
during the 18F-fluoride PET and CT angiogram scanning visits including 
administration of the beta blockade, glyceryl trinitrate, contrast agent and 
radiation exposure. Patients attending the scanning visits will be asked to 
contact the research team if they experience any untoward effects within 48 
hours of the scans. Only AEs/SAEs experienced during the scan and 
selfreported within 48 hours of the PET and /or CT scan will be recorded in the 
AE log and SAE form, respectively. Any AEs or SAEs out with the above 

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definitions will not be recorded during the study although data on 
hospitalisations and deaths will be collected for long-term outcomes.  
11.3 RECORDING AEs AND SAEs  
  
Any pre-existing medical conditions (i.e. existed prior to informed consent) are 
not adverse events but should be recorded as medical history in the CRF. A 
pre-existing medical condition should only be recorded as an adverse event if 
the condition worsens during the study. The event term should clearly document 
that the condition has worsened from baseline.  
  
When an AE/SAE occurs, it is the responsibility of the Investigator to review all 
documentation (e.g. hospital notes, laboratory and diagnostic reports) related 
to the event.  The Investigator will then record all relevant information in the 
CRF and on the SAE form (if the AE meets the criteria of serious).  
Information to be collected includes dose, type of event, onset date, Investigator 
assessment of severity and causality, date of resolution as well as treatment 
required, investigations needed and outcome.    
11.4 ASSESSMENT OF AEs AND SAEs  
Seriousness, causality, severity and expectedness will be assessed by the 
Principal Investigator. Cases that are considered serious, possibly, probably or 
definitely related to IMP and unexpected (i.e. SUSARs) will be unblinded.   
The Investigator is responsible for assessing each AE.    
The Chief Investigator may not downgrade an event that has been assessed by 
an Investigator as an SAE or SUSAR, but can upgrade an AE to an SAE, SAR 
or SUSAR if appropriate.  
11.4.1 Assessment of Seriousness  
The Investigator will make an assessment of seriousness as defined in Section 
11.1.  
11.4.2 Assessment of Causality  
The Investigator will make an assessment of whether the AE/SAE is likely to be 
related to the IMP according to the definitions below.    
• 
Unrelated: where an event is not considered to be related to the IMP.  
• 
Possibly Related: The nature of the event, the underlying medical 
condition, concomitant medication or temporal relationship make it 
possible that the AE has a causal relationship to the study drug. The 
assessment of causality will be made against the reference safety 
information found in the Investigator’s Brochure.   
Where non Investigational Medicinal Products (NIMPs) e.g. rescue/escape 
drugs are given:  if the AE is considered to be related to an interaction between 
the IMP and the NIMP, or where the AE might be linked to either the IMP or the 
NIMP but cannot be clearly attributed to either one of these, the event will be 
considered as an AR. Alternative causes such as natural history of the 
underlying disease, other risk factors and the temporal relationship of the event 

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to the treatment should be considered and investigated. The blind should not 
be broken for the purpose of making this assessment.   
11.4.3 Assessment of Expectedness  
If an event is judged to be an AR, the evaluation of expectedness will be made 
based on knowledge of the reaction and the relevant product information 
documented in the IB.  
The event may be classed as either:  
Expected: the AR is consistent with the toxicity of the IMP listed in the IB. 
Unexpected: the AR is not consistent with the toxicity in the IB.  
11.4.4 Assessment of Severity  
The Investigator will make an assessment of severity for each AE/SAE and 
record this on the CRF or SAE form according to one of the following categories: 
Mild: an event that is easily tolerated by the participant, causing minimal 
discomfort and not interfering with everyday activities.  
Moderate: an event that is sufficiently discomforting to interfere with normal 
everyday activities.  
Severe: an event that prevents normal everyday activities.  
Note: the term ‘severe’, used to describe the intensity, should not be confused 
with ‘serious’ which is a regulatory definition based on participant/event 
outcome or action criteria.  For example, a headache may be severe but not 
serious, while a minor stroke is serious but may not be severe.  
11.5 REPORTING OF SAEs/SARs/SUSARs  
Once the Investigator becomes aware that an SAE has occurred in a study 
participant, the information will be reported to the ACCORD Research 
Governance & QA Office immediately or within 24 hours. If the Investigator 
does not have all information regarding an SAE, they should not wait for this 
additional information before notifying ACCORD.  The SAE report form can be 
updated when the additional information is received.  
The SAE report will provide an assessment of causality and expectedness at 
the time of the initial report to ACCORD according to Sections 11.4.2, 
Assessment of Causality and 11.4.3, Assessment of Expectedness. The SAE 
form will be transmitted by fax to ACCORD on +44 (0)131 242 9447 or may be 
transmitted by hand to the office or submitted via email to safety@accord.scot. 
Only forms in a pdf format will be accepted by ACCORD via email.  
Where missing information has not been sent to ACCORD after an initial report, 
ACCORD will contact the investigator and request the missing information.  All 
reports faxed to ACCORD and any follow up information will be retained by the 
Investigator in the Investigator Site File (ISF).  
11.6 REGULATORY REPORTING REQUIREMENTS  
The ACCORD Research Governance & QA Office is responsible for 
pharmacovigilance reporting on behalf of the co-sponsors (Edinburgh 
University and NHS Lothian).  

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The ACCORD Research Governance & QA Office has a legal responsibility to 
notify the regulatory competent authority and relevant ethics committee 
(Research Ethics Committee (REC) that approved the trial).  Fatal or life 
threatening SUSARs will be reported no later than 7 calendar days and all other 
SUSARs will be reported no later than 15 calendar days after ACCORD is first 
aware of the reaction.    
ACCORD will inform Investigators at participating sites of all SUSARs and any 
other arising safety information.  
An Annual Safety Report/Development Safety Update Report will be submitted, 
by ACCORD, to the regulatory authorities and RECs listing all SARs and 
SUSARs.  
11.7 FOLLOW UP PROCEDURES  
After initially recording an AE or recording and reporting an SAE, the 
Investigator should make every effort to follow each event until a final outcome 
can be recorded or reported as necessary. Follow up information on an SAE 
will be reported to the ACCORD office.  
If, after follow-up, resolution of an event cannot be established, an explanation 
should be recorded on the CRF, AE log or SAE form.  
12 PREGNANCY  
Women who are pregnant, breastfeeding or of child-bearing potential (women 
who have experienced menarche, are pre-menopausal and have not been 
sterilised) will not be enrolled into the trial.  
13 TRIAL MANAGEMENT AND OVERSIGHT ARRANGEMENTS  
13.1 TRIAL MANAGEMENT GROUP  
The trial will be coordinated by a Project Management Group, consisting of the 
grant holders (Chief Investigator and Principal Investigator in Edinburgh) and 
site leads.  
The Principal Investigator will oversee the study and will be accountable to the 
Chief Investigator.  The Principal Investigator will be responsible for checking 
the CRFs for completeness, plausibility and consistency.  Any queries will be 
resolved by the Investigator or delegated member of the trial team.   
A Delegation Log will be prepared for each site, detailing the responsibilities of 
each member of staff working on the trial.    
13.2 TRIAL STEERING COMMITTEE  
A Trial Steering Committee (TSC) will be established to oversee the conduct 
and progress of the trial.  Contact details of the TSC are detailed in a separate 
TSC charter. The review of SAEs will be added to the TSC agenda to ensure 
that appropriate action is taken if any safety issues arise.    

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13.3 DATA MONITORING COMMITTEE  
This is an observational open label clinical cohort study involving an established 
radiotracer with over 40 years of experience. The result of the 18F-fluoride 
positron emission tomography and computed tomography coronary angiogram 
will not be fed back to the clinical team responsible for the patients care (except 
for clinically important incidental findings). For this reason, an independent Data 
Monitoring Committee (DMC) will not be convened for this study and all study 
serious adverse events will be reported to the sponsor and will be discussed by 
the TSC.  
13.4 INSPECTION OF RECORDS  
Investigators and institutions involved in the study will permit trial related 
monitoring and audits on behalf of the sponsor, REC review, and regulatory 
inspection(s).  In the event of an audit or monitoring, the Investigator agrees to 
allow the representatives of the sponsor direct access to all study records and 
source documentation. In the event of regulatory inspection, the Investigator 
agrees to allow inspectors direct access to all study records and source 
documentation.  
13.5 RISK ASSESSMENT  
A study specific risk assessment has been performed by representatives of the 
co-sponsors, ACCORD monitors and the QA group, in accordance with  
ACCORD governance and sponsorship SOPs. Input has been sought from the 
Chief Investigator or designee. The outcomes of the risk assessment were the 
basis from which monitoring plans and audit plans were created. The risk 
assessment outcomes also indicated which risk adaptions (delete if no 
adaptations were possible) could be incorporated into to trial design.  
13.6 BENEFIT/RISK BALANCE  
13.6.1 Benefits  
Patients may also benefit from additional procedures and investigations that 
they will undergo as part of the study. This will include closer medical 
supervision and non-invasive imaging investigations that may identify important 
incidental findings.  
13.6.2 Risks  
There are some potential hazards of the non-invasive investigations that we will 
perform as part of the trial. The main issues relate to exposure to ionising 
radiation and contrast agent administration. We have a well-developed protocol 
for cardiac positron emission and computed tomography imaging that 
minimises radiation exposure and has clear procedures for managing adverse 
contrast reactions. We anticipate that the total research protocol dose (TPRD) 
radiation exposure not exceed 34 mSv. The TRPD includes radiation exposure 
from both the positron emission tomography and computed tomography scans 
combined. The estimated associated risk of developing fatal cancer is 
proportional to dose.  Using a risk of 5% per Sv [ARSAC Notes] in a healthy 

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population in this age group the estimated associated risk of developing fatal 
cancer as a result of this exposure is in the region 1 in 650.  This risk can be 
classified as moderate. It is likely that in a population of any patients in the age 
group 50+ the cancer risk is lower than 5% per Sv.  For comparison the average 
annual background radiation dose arising from natural sources of ionising 
radiation in the environment in the UK is 2.2 mSv.  The TRPD of 34 mSv 
incurred in this study is approximately 15 times annual background radiation 
from natural sources.   This can be compared with other commonly used 
cardiovascular imaging techniques, such as nucleotide myocardial perfusion 
imaging (15-20 mSv) and diagnostic coronary angiography (7 mSv) [Einstein et 
al, 2007].   
The risks of exposure to the contrast medium include allergic reactions and 
impairment of kidney function. Amongst patients with moderate-to-severe 
chronic kidney disease, there is a 2-4% risk of kidney impairment after 
computed tomography angiography [Barrett et al, 2006]. The risk of contrast 
exposure in this study will be minimised by exclusion of high-risk patients who 
have significant kidney disease (estimated glomerular filtration rate <30 
mL/min/1.73m2).  
13.7 STUDY MONITORING AND AUDIT  
ACCORD clinical trial monitors, or designees, will perform monitoring activities 
in accordance with the study monitoring plan. This will involve on-site visits and 
central monitoring activities as necessary (delete where not required). 
ACCORD QA personnel, or designees, will perform study audits in accordance 
with the study audit plan. This will involve investigator site audits, study 
management audits and facility (including 3rd parties) audits as necessary 
(delete where not required).   
14 GOOD CLINICAL PRACTICE  
14.1 ETHICAL CONDUCT  
The study will be conducted in accordance with the principles of the 
International Conference on Harmonisation Tripartite Guideline for Good 
Clinical Practice (ICH GCP).  
A favorable ethical opinion will be obtained from the appropriate REC and local 
R&D approval will be obtained prior to commencement of the study.  
14.2 REGULATORY COMPLIANCE  
The study will not commence until a Clinical Trial Authorisation (CTA) is 
obtained from the appropriate Regulatory Authority. The protocol and study 
conduct will comply with the Medicines for Human Use (Clinical Trials) 
Regulations 2004, as amended.  
14.3 INVESTIGATOR RESPONSIBILITIES  
The Investigator is responsible for the overall conduct of the study at the site 
and compliance with the protocol and any protocol amendments.  In accordance 

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with the principles of ICH GCP, the following areas listed in this section are also 
the responsibility of the Investigator.  Responsibilities may be delegated to an 
appropriate member of study site staff.  
14.3.1 Informed Consent  
The Investigator is responsible for ensuring informed consent is obtained before 
any protocol specific procedures are carried out.  The decision of a participant 
to participate in clinical research is voluntary and should be based on a clear 
understanding of what is involved.  
Participants must receive adequate oral and written information – appropriate 
Participant Information and Informed Consent Forms will be provided.  The oral 
explanation to the participant will be performed by the Investigator or qualified 
delegated person and must cover all the elements specified in the Participant 
Information Sheet and Consent Form.  
The participant must be given every opportunity to clarify any points they do not 
understand and, if necessary, ask for more information.  The participant must 
be given sufficient time to consider the information provided.  It should be 
emphasised that the participant may withdraw their consent to participate at any 
time without loss of benefits to which they otherwise would be entitled. The 
participant will be informed and agree to their medical records being inspected 
by regulatory authorities and representatives of the sponsor(s) but understand 
that their name will not be disclosed outside the hospital.  
The Investigator or delegated member of the trial team and the participant will 
sign and date the Informed Consent Form(s) to confirm that consent has been 
obtained.  The participant will receive a copy of this document and a copy filed 
in the Investigator Site File (ISF) and participant’s medical notes.  
14.3.2 Study Site Staff  
The Investigator must be familiar with the IMP, protocol and the study 
requirements.  It is the Investigator’s responsibility to ensure that all staff 
assisting with the study are adequately informed about the IMP, protocol and 
their trial related duties.  
14.3.3 Data Recording  
The Principal Investigator is responsible for the quality of the data recorded in 
the CRF at each Investigator Site. The source data plan identifies which source 
data correspond to CRF data and states which data are recorded directly into 
the CRF.  
14.3.4 Investigator Documentation  
Prior to beginning the study, each Investigator will be asked to provide particular 
essential documents to the ACCORD Research Governance & QA Office, 
including but not limited to:  
• 
An original signed Investigator’s Declaration (as part of the Clinical Trial 
Agreement documents);  
• 
Curriculum vitae (CV) signed and dated by the Investigator indicating 
that it is accurate and current.  

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The ACCORD Research Governance & QA Office will ensure all other 
documents required by ICH GCP are retained in a Trial Master File (TMF), 
where required, and that appropriate documentation is available in local ISFs.  
14.3.5 GCP Training  
All study staff will hold evidence of GCP training where appropriate.   
14.3.6 Confidentiality  
All laboratory specimens, evaluation forms, reports, and other records must be 
identified in a manner designed to maintain participant confidentiality.  All 
records must be kept in a secure storage area with limited access.  Identifiable 
clinical information will not be released without the written permission of the 
participant.  The Investigator and study site staff involved with this study may 
not disclose or use for any purpose other than performance of the study, any 
identifiable data, record, or other unpublished, confidential information 
disclosed to those individuals for the purpose of the study.  Prior written 
agreement from the sponsor or its designee must be obtained for the disclosure 
of any said confidential information to other parties.  
14.3.7 Data Protection  
All Investigators and study site staff involved with this study must comply with 
the requirements of the appropriate data protection legislation (including where 
applicable the General Data Protection Regulation with regard to the collection, 
storage, processing and disclosure of personal information. Access to personal 
information will be restricted to individuals from the research team treating the 
participants, representatives of the sponsor(s) and representatives of regulatory 
authorities.  
Computers used to collate the data will have limited access measures via user 
names and passwords.  
Published results will not contain any personal data that could allow 
identification of individual participants.  
15 STUDY CONDUCT RESPONSIBILITIES  
15.1 PROTOCOL AMENDMENTS  
Any changes in research activity, except those necessary to remove an 
apparent, immediate hazard to the participant in the case of an urgent safety 
measure, must be reviewed and approved by the Chief Investigator.   
Amendments to the protocol must be submitted in writing to the appropriate 
REC, Regulatory Authority and local R&D for approval prior to participants 
being enrolled into an amended protocol.  
15.2 PROTOCOL VIOLATIONS AND DEVIATIONS  
Prospective protocol deviations, i.e. protocol waivers, will not be approved by 
the sponsors and therefore will not be implemented, except where necessary 
to eliminate an immediate hazard to study participants. If this necessitates a 

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subsequent protocol amendment, this should be submitted to the REC, 
Regulatory Authority and local R&D for review and approval if appropriate. 
Protocol deviations will be recorded in a protocol deviation log and logs will be 
submitted to the sponsors every 3 months. Each protocol violation will be 
reported to the sponsor within 3 days of becoming aware of the violation. 
Deviation logs / violation forms will be transmitted via email to QA@accord.scot 
Only forms in a pdf format will be accepted by ACCORD via email. Forms may 
also be sent by fax to ACCORD on +44 (0)131 242 9447 or may be submitted 
by hand to the office. Where missing information has not been sent to ACCORD 
after an initial report, ACCORD will contact the Investigator and request the 
missing information. The Investigator must respond to these requests in a timely 
manner  
15.3 SERIOUS BREACH REQUIREMENTS  
A serious breach is a breach which is likely to effect to a significant degree: (a) 
the safety or physical or mental integrity of the participants of the trial; or (b) 
the scientific value of the trial.  
If a potential serious breach is identified by the Chief investigator, Principal 
Investigator or delegates, the co-sponsors (QA@accord.scot) must be notified 
within 24 hours.  It is the responsibility of the co-sponsors to assess the impact 
of the breach on the scientific value of the trial, to determine whether the 
incident constitutes a serious breach and report to regulatory authorities and 
research ethics committees as necessary.   
15.4 STUDY RECORD RETENTION  
All study documentation will be kept for a minimum of 5 years from the protocol 
defined end of study point. When the minimum retention period has elapsed, 
study documentation will not be destroyed without permission from the sponsor.  
15.5 END OF STUDY  
It is anticipated that the study will last 6 years: four years for recruitment and 
two years of follow-up. The end of study is defined as the last participant’s last 
visit.    
The Investigators and/or the trial steering committee and/or the co-sponsor(s) 
have the right at any time to terminate the study for clinical or administrative 
reasons.   
The end of the study will be reported to the REC, Regulatory Authority, R&D 
Office(s) and co-sponsors within 90 days, or 15 days if the study is terminated 
prematurely. The Investigators will inform participants of the premature study 
closure and ensure that the appropriate follow up is arranged for all participants 
involved. End of study notification will be reported to the co-sponsors via email 
to resgov@ed.ac.uk.  
In accordance with ACCORD SOP CR011, a Clinical Study Report (CSR) will 
be provided to the Sponsor (QA@accord.scot) and REC within 1 year of the 
end of the study.  
Upon completion of the study, the Investigator will upload clinical trial results 
onto the EudraCT database on behalf of the Sponsor.   

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15.6 INSURANCE AND INDEMNITY  
The co-sponsors are responsible for ensuring proper provision has been made 
for insurance or indemnity to cover their liability and the liability of the Chief 
Investigator and staff.  
The following arrangements are in place to fulfil the co-sponsors' 
responsibilities:  
• 
The Protocol has been designed by the Chief Investigator and 
researchers employed by the University and collaborators.  The 
University 
has 
insurance 
in 
place 
(which 
includes 
no-fault 
compensation) for negligent harm caused by poor protocol design by 
the Chief Investigator and researchers employed by the University.  
• 
Sites participating in the study will be liable for clinical negligence and 
other negligent harm to individuals taking part in the study and covered 
by the duty of care owed to them by the sites concerned.  The 
cosponsors require individual sites participating in the study to arrange 
for their own insurance or indemnity in respect of these liabilities.  
• 
Sites which are part of the United Kingdom's Nation Health Service will 
have the benefit of NHS Indemnity.  
• 
Sites out with the United Kingdom will be responsible for arranging their 
own indemnity or insurance for their participation in the study, as well 
as for compliance with local law applicable to their participation in the 
study.  
• 
The manufacturer supplying IMP has accepted limited liability related 
to the manufacturing and original packaging of the study drug and to 
the losses, damages, claims or liabilities incurred by study participants 
based on known or unknown Adverse Events which arise out of the 
manufacturing and original packaging of the study drug, but not where 
there is any modification to the study drug (including without limitation 
re-packaging and blinding).  
16 REPORTING, PUBLICATIONS AND NOTIFICATION OF RESULTS  
16.1 AUTHORSHIP POLICY  
Ownership of the data arising from this study resides with the study team.  On 
completion of the study, the study data will be analysed and tabulated, and a 
clinical study report will be prepared in accordance with appropriate regulatory 
guidelines.   
16.2 PUBLICATION  
The clinical study report will be used for publication and presentation at scientific 
meetings. Investigators have the right to publish orally or in writing the results 
of the study.  
Summaries of results will also be made available to Investigators for 
dissemination within their clinics (where appropriate and according to their 
discretion).  

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16.3 PEER REVIEW  
The study has undergone national and international peer review by the  
Wellcome Trust. There is additional peer review from the Trial Steering 
Committee and the Edinburgh Clinical Trials Unit as well as the study sponsors.  
  
  
 

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17 REFERENCES  
ARSAC  Notes  for  Guidance  on  the  Clinical  Administration  of  
Radiopharmaceuticals and Use of Sealed Radioactive Sources -March 2006 
(updated 2014) Appendix VII  
Barrett BJ, Katzberg RW, Thomsen HS, Chen N, Sahani D, Soulez G, Heiken 
JP, Lepanto L, Ni ZH, Ni ZH, Nelson R. Contrast-induced nephropathy in 
patients with chronic kidney disease undergoing computed tomography: a 
double-blind comparison of iodixanol and iopamidol. Invest Radiol. 
2006;41:815-821.  
Davies M. The pathophysiology of acute coronary syndromes. Heart. 
2000;83:361-6.  
Detrano R, Guerci AD, Carr JJ, et al. Coronary calcium as a predictor of 
coronary events in four racial or ethnic groups. N Engl J Med 
2008;358:13361345.  
Dweck MR, Chow MW, Joshi NV, Williams MC, Jones C, Fletcher AM, 
Richardson H, White A, McKillop G, van Beek EJ, Boon NA, Rudd JH, Newby 
DE. Coronary arterial 18f-sodium fluoride uptake: A novel marker of plaque 
biology. J Am Coll Cardiol. 2012b;59:1539-1548  
Dweck MR, Jones C, Joshi NV, Fletcher AM, Richardson H, White A, Marsden 
M, Pessotto R, Clark JC, Wallace WA, Salter DM, McKillop G, van Beek EJ, 
Boon NA, Rudd JH, Newby DE. Assessment of valvular calcification and 
inflammation by positron emission tomography in patients with aortic stenosis. 
Circulation. 2012a;125:76-86  
Dweck MR, Khaw HJ, Sng GK, Luo EL, Baird A, Williams MC, Makiello P, 
Mirsadraee S, Joshi NV, van Beek EJ, Boon NA, Rudd JH, Newby DE. Aortic 
stenosis, atherosclerosis, and skeletal bone: Is there a common link with 
calcification and inflammation? Eur Heart J. 2013;34:1567-1574.  
Einstein AJ, Moser KW, Thompson RC, Cerqueira MD, Henzlova MJ. Radiation 
dose 
to 
patients 
from 
cardiac 
diagnostic 
imaging. 
Circulation. 
2007;116:12901305.  
Joshi NV, Vesey AT, Williams MC, Shah ASV, Calvert PA, Craighead FHM, 
Yeoh SE, Wallace W, Salter D, Fletcher AM, van Beek EJR, Flapan AD, Uren 
NG, Behan MWH, Cruden NLM, Mills NL, Fox KAA, Rudd JHF, Marc R Dweck 
MR, Newby DE. 18F-Fluoride positron emission tomography identifies ruptured 
and high-risk coronary atherosclerotic plaques. Lancet 2014;383:705-713.  
Kwiecinski J, Cadet S, Daghem M, Lassen ML, Dey D, Dweck MR, Berman DS, 
Newby DE, Slomka PJ. Whole-vessel coronary (18)F-sodium fluoride PET for 
assessment of the global coronary microcalcification burden. Eur J Nucl Med 
Mol Imaging. 2020a;47:1736-1745.  
Kwiecinski J, Tzolos E, Adamson PD, Cadet S, Moss AJ, Joshi N, Williams MC, 
van Beek EJR, Dey D, Berman DS, Newby DE, Slomka PJ, Dweck MR. 
Coronary (18)F-Sodium Fluoride Uptake Predicts Outcomes in Patients With 
Coronary Artery Disease. J Am Coll Cardiol. 2020b;75:3061-3074.  Mann J, 
Davies MJ. Mechanisms of progression in native coronary artery disease: role 
of healed plaque disruption. Heart. 1999;82:265-8.  

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Mills NL, Churchhouse AM, Lee KK, et al. Implementation of a sensitive troponin 
I assay and risk of recurrent myocardial infarction and death in patients with 
suspected acute coronary syndrome. JAMA 2011;305:1210-16.  
Mills NL, Lee KK, McAllister DA, Churchhouse AMD, MacLeod M, Stoddard M, 
Walker S, Denvir MA, Fox KAA, Newby DE. Implications of lowering the 
threshold of plasma troponin in the diagnosis of myocardial infarction: a cohort 
study. BMJ 2012;344:e1533.  
Omland T, Pfeffer MA, Solomon SD, de Lemos JA, Rosjo H, Saltyte Benth J, 
Maggioni A, Domanski MJ, Rouleau JL, Sabatine MS, Braunwald E. Prognostic 
value of cardiac troponin i measured with a highly sensitive assay in patients 
with stable coronary artery disease. J Am Coll Cardiol. 2013;61:1240-1249 
Rioufol G, Gilard M, Finet G, Ginon I, Boschat J, André-Fouët X. Evolution of 
spontaneous atherosclerotic plaque rupture with medical therapy: long-term 
follow-up with intravascular ultrasound. Circulation. 2004;110:2875-80. Shah 
ASV, Griffiths M, Lee KK, McAllister, DA, Hunter AL, Cruikshank A, Alan Reid 
A, Stoddart M, Strachan F, Walker S, Collinson PO,Apple FS, Gray AJ, Fox 
KAA, Newby DE, Mills NL. High-sensitivity cardiac troponin and the under 
diagnosis of myocardial infarction in women. ESC Hotline 2013.  
Thomas GS, Haraszti RA. A new frontier in atherosclerotic coronary imaging. 
Lancet. 2014;383:674-5.   
Thompson RC, Allam AH, Lombardi GP, Wann LS, Sutherland ML, Sutherland 
JD, Soliman MA, Frohlich B, Mininberg DT, Monge JM, Vallodolid CM, Cox SL, 
Abd el-Maksoud G, Badr I, Miyamoto MI, el-Halim Nur el-Din A, Narula J, Finch 
CE, Thomas GS. Atherosclerosis across 4000 years of human history: the 
Horus study of four ancient populations. Lancet. 2013;381:1211-22.  
Tzolos E, Kwiecinski J, Lassen ML, Cadet S, Adamson PD, Moss AJ, Joshi N,  
Williams MC, van Beek EJR, Dey D, Berman DS, Dweck MR, Newby DE, 
Slomka PJ. Observer repeatability and interscan reproducibility of 18F-sodium 
fluoride coronary microcalcification activity. J Nucl Cardiol. 2020; in press. doi:  
10.1007/s12350-020-02221-1.   
  
 
 
 
 
 
 
 
 
 
 
  
   

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FIGURE 1.  Focal 18F-Fluoride Uptake in Patients with Myocardial 
Infarction and Stable Angina  
Patient with acute ST-segment elevation myocardial infarction with (A) proximal 
occlusion (red arrow) of the left anterior descending artery on invasive coronary 
angiography and (B) intense focal 18F-fluoride uptake (yellow-red) at the site 
of the culprit plaque (red arrow) on the combined positron emission and 
computed tomogram.   
Patient with anterior non-ST-segment elevation myocardial infarction with (C) 
culprit (red arrow; left anterior descending artery) and bystander non-culprit 
(white arrow; circumflex artery) lesions on invasive coronary angiography that 
were both stented during the index admission. Only the culprit lesion had 
increased 18F-fluoride uptake on combined positron emission and computed 
tomography (D) following percutaneous coronary intervention.   
  
 
 
 
 
 
 

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FIGURE 2.  Patients with Stable Angina and 18F-Fluoride Uptake  
Representative examples for 18F-fluoride uptake in patients with stable angina. 
Panels A-D, computed tomography coronary angiograms; panels E-H, 
18Ffluoride positron emission tomograms; and panels I-L, fused positron 
emission tomograms and computed tomography coronary angiograms.   
 
  
  
 
 
 
 
 
 
  
F u s e d P E T / C T C A 
V H - I V U S 
I 
A 
K 
C 
G 
D 
L 
H 
E 
B 
F 
J 

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FIGURE 3. Patient Flow Chart  
  
  
 
   
Patients with myocardial infarction and  
multivessel disease (n=700)   
PRE18FFIR - IMAGE study   
Baseline 18F - Fluoride PET and  
CTCA   
PRE18FFIR - REPRO Study   
PRE18FFIR - TIME Study   
Telephone Interview at 1year and  
periodically   thereafter until study  
completion   
Ongoing periodic review of electronic Health Records  
for 5 years following study completion 
  
Follow - up CTCA at 2 years   

   
 
Statistical Analysis Plan  PRE18FFIR  
 
Version No  
V1.0  
 
Date Finalised   
23 Mar 2022  
  
Page 1 of 9 ST004-SAP Template /v4.0/25 Mar 2021  
  
 
  
PRE18FFIR  
Prediction of Recurrent Events with 18F-Fluoride to Identify Rupture and High-risk Coronary Artery 
Plaques in Patients with Myocardial Infarction  
  
Statistical Analysis Plan  
  
Version No  
1.0  
Date Finalised   
23 March 2022  
Author(s)  
Lumine Na (to Feb 2016), Steff Lewis (after Feb 2016)  
Chief Investigator  
Dave Newby  
Chief Investigator Email Address  d.e.newby@ed.ac.uk  
  
  
Signatures  
 
Trial Statistician:  
  
  
  
Date:  
Chief Investigator:  
  
  
  
Date:  
  
  
Document Control  
 
 
Version No  
Date  
Summary of Revisions  
1.0  
23 March  
2022  
Finalised SAP created  
Draft versions prior to 
official numbering of 
V1.0  
09 Feb 2016  
Draft SAP created, and edited following comments from 
chief investigator and trial statistician  
  
  
  

   
 
Statistical Analysis Plan  PRE18FFIR  
 
Version No  
V1.0  
 
Date Finalised   
23 Mar 2022  
  
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Table of Contents  
List of Abbreviations ........................................................................................................................... 3 
1.  Introduction ................................................................................................................................... 4 
2.  Statistical Methods section from the protocol .............................................................................. 4 
3.  Overall Statistical Principles ........................................................................................................... 5 
4.  Analysis Populations ...................................................................................................................... 5 
5.  List of Analyses ............................................................................................................................... 5 
5.1  Recruitment and retention .......................................................................................................... 5 
5.2  Baseline characteristics................................................................................................................ 6 
5.3  Primary endpoint ......................................................................................................................... 7 
5.4  Secondary endpoints ................................................................................................................... 8 
5.5  Safety analyses ............................................................................................................................. 9 
5.6.  Deviations, Violations ................................................................................................................. 9 
6.  Validation and QC .......................................................................................................................... 9 
7.  Data sharing ................................................................................................................................. 10 
8.  References ................................................................................................................................... 10 
 
  
  
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 

   
 
Statistical Analysis Plan  PRE18FFIR  
 
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List of Abbreviations   
  
Abbreviation  
Full name   
18F-fluoride  
18F-sodium fluoride  
ACE  
Angiotensin Converting Enzyme  
ARB  
Angiotensin Receptor Blocker  
CABG  
Coronary Artery Bypass Graft  
CI  
Confidence Interval  
CMA  
Coronary Microcalcification Activity  
CMA status  
Binary classification of whether or not a participant has coronary 
microcalcification activity  
CT  
Computed Tomography  
CONSORT  
Consolidated Standards of Reporting Trials  
ECTU  
Edinburgh Clinical Trials Unit  
GRACE  
Global Registry of Acute Coronary Events  
IMP  
Investigational Medicinal Product  
LMS  
Left Main Stem coronary artery  
MBq  
Megabecquerel  
MedDRA  
Medical Dictionary for Regulatory Activities  
MI  
Myocardial Infarction  
mSv  
Millisievert  
N  
Number of participants with an observation  
NIMP  
Non-Investigational Medicinal Product  
NSTEMI  
Non-ST Elevation Myocardial Infarction  
PCI  
Percutaneous Coronary Intervention  
PET  
Positron Emission Tomography  
PRE18FFIR  
Trial acronym for ‘Prediction of Recurrent Events with 18F-Fluoride to Identify 
Ruptured and High-risk Coronary Artery Plaques with Myocardial Infarction’  
Q1  
Lower quartile  
Q3  
Upper quartile  
SAP  
Statistical Analysis Plan  
SD  
Standard Deviation  
STEMI  
ST Elevation Myocardial Infarction  
TIA  
Transient Ischaemic Attack  
  
  

   
 
Statistical Analysis Plan  PRE18FFIR  
 
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1.  
Introduction  
This document details the criteria to be used for the definition of the analysis populations and the 
statistical methodology for analysis for the PRE18FFIR trial. This SAP covers the main PRE18FFIR 
protocol. It does not relate to the PRE18FFIR substudies that are covered by separate protocols – these 
will not be analysed by the ECTU statistics team. In addition, this SAP does not cover the exploratory 
objectives in the protocol – these will not be analysed by the ECTU statistics team.  
PRE18FFIR is a prospective multi-centre observational cohort study to investigate the associations 
between coronary 18F-fluoride uptake and disease progression and clinical outcomes, in patients with 
recent myocardial infarction and multi-vessel coronary artery disease.  All participants receive the IMP, 
18F-sodium fluoride (18F-fluoride), and undergo 18F-fluoride positron emission tomography to 
determine their level of 18F-fluoride uptake. The aim is to recruit 700 participants.  
This document has been compiled according to the Edinburgh Clinical Trials Unit (ECTU) standard 
operating procedure “Statistical Analysis Plans” ECTU_ST_04 and has been written based on 
information contained in the PRE18FFIR study protocol version 11.0, dated 24 February 2022.  
  
2.  
Statistical Methods section from the protocol  
The primary analysis will be performed by calculating Kaplan-Meier estimates of the ‘survival’ curves 
for the time to first event (cardiac death, recurrent myocardial infarction, or unscheduled coronary 
revascularisation) during follow up, and comparing these estimates for participants with and without 
coronary microcalcification activity using a log-rank test. The results will be expressed as the estimated 
hazard ratio with the corresponding 95% confidence interval and p value. Participants who die during 
follow-up whose deaths are not classified as cardiac deaths will be censored in the above analysis at 
the time of death.   
Since 18F-fluoride uptake is a continuous measure, a potentially more sensitive analysis will explore the 
relationship between uptake and the risk of suffering an event (cardiac death, recurrent myocardial 
infarction, or unscheduled coronary revascularisation). This will be performed using the Cox 
proportional hazards regression model, with (transformed) 18F-fluoride uptake as the only covariate. 
A range of potential transformations of the 18F-fluoride uptake will be explored, including fractional 
polynomials, to find an optimal fit to the data.  
Once an appropriate transformation for the 18F-fluoride uptake has been identified (as above), the 
Global Registry of Acute Coronary Events (GRACE) score will be added into the Cox proportional hazards 
model, to assess the incremental prognostic value of 18F-fluoride over and above conventional clinical 
risk markers.  
  
There are 3 secondary endpoints in the protocol that will be analysed later (not by ECTU), and will not 
form part of this SAP:   
  
• 
18F-fluoride uptake localisation to culprit plaque causing the index myocardial infarction  
• 
Territory of subsequent myocardial reinfarction  
• 
Coronary artery plaque progression by computed tomography coronary angiography. Plaque 
volume, composition and calcification  
  

   
 
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3.  
Overall Statistical Principles   
In general terms, categorical data will be presented using counts (N) and percentages (%), whilst 
continuous variables will be presented using the mean, median, standard deviation (SD), minimum, 
maximum, lower and upper quartiles [Q1, Q3], and number of participants with an observation (N). 
Data will be split by time point where applicable.   
In general, analyses will be unadjusted, unless otherwise specified.  
All applicable statistical tests will be 2-sided and will be performed using a 5% significance level. 95% 
(2-sided) confidence intervals (CIs) will be presented. All analyses are seeking to show a difference, 
rather than equivalence or non-inferiority.    
All analyses and data manipulations will be carried out using the most up to date version of SAS 
available [1].  
There are no pre-planned subgroup analyses.  
Note: Throughout this document, “study entry” is defined as the date a participant had the baseline 
PET-CT scan as captured on the baseline visit case report form. “CMA status” is used to denote the 
binary classification of whether or not a participant had coronary microcalcification activity. Those 
participants with CMA>0 are defined as having coronary microcalcification activity, and those with 
CMA=0 as not having any coronary microcalcification activity.   
Note: Due to Covid-19, some follow ups at two years will have the imaging visit and the rest of the 
follow up done at different times.  The database only captures one date.  This should be for the 
nonimaging follow-up.  It should not be assumed that two year follow up dates relate to the imaging 
visits.  
  
4.  
Analysis Populations   
Scanned population  
This population will include all patients who had the baseline PET-CT scan completed.   
Analysis population  
This population will include all patients who had the baseline PET-CT scan and CMA status available 
from analysis of scan images.   
All analyses will be performed on the analysis population unless otherwise specified.    
Safety population  
This population will include all patients who received the radioactive tracer.   
  
5.  
List of Analyses   
5.1  
Recruitment and retention  
The data to construct a CONSORT flow chart will be provided, as far as they are available in the study 
database. We will aim to tabulate: number of patients who were approached to participate in the 
study; number of patients who were excluded (with a tabulation of reasons for exclusion); number of 
patients who had the screening visit; number of patients who were excluded (with a tabulation of 
reasons for exclusion); number of patients who attended the baseline visit and received the 

   
 
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radioactive tracer [18F-fluoride]; number of patients who received the radioactive tracer and the 
baseline scan was not started, or started but aborted (with tabulation of reasons); number of patients 
who had baseline scan completed ; number of participants who withdrew consent including to use of 
existing data; number of participants with follow-up completed or who died, or had follow-up 
discontinued prematurely (with tabulation of reasons for premature discontinuation); ; total 
personyears of follow-up during the study, before Covid-19, and during Covid-19; number of 
participants included in analysis of primary outcome;  number of participants excluded from analysis 
of primary outcome (with tabulation of reasons).  Median, lower and upper quartile, minimum and 
maximum for duration of follow-up (overall and split by CMA status).  
The statistical report will provide for the scanned population, not split by CMA status, a graph of 
cumulative number of patients recruited into the study over time and the dates the first and last 
patients were recruited; the number of study sites that recruited patients; a tabulation of the number 
of patients recruited by each study site.  
  
5.2  
Baseline characteristics  
The following baseline characteristics will be tabulated by CMA status, and overall. No formal 
statistical testing will be performed.  
Demographics  
a. Sex (Male/Female)  
b. Age (years) and (18-64, 65-84, ≥85)  
c. 
Body mass index (kg/m2)  
Cardiovascular Risk Factors  
d. Smoking status (Never/Ex-smoker/Current)  
e. Hypertension or receiving treatment for hypertension (Yes/No)  
f. 
High cholesterol (>5 mmol/L) or on cholesterol lowering treatment prior to admission (Yes/No) 
g. Diabetes mellitus (Yes/No) if Yes then type (Type 1/Type 2)  
Cardiovascular History  
h. Previous diagnosis of ischaemic heart disease prior to admission (Yes/No)  
i. 
Documented history of myocardial infarction prior to admission (Yes/No)  
j. 
Previous PCI prior to admission (Yes/No)   
k. 
Previous CABG (Yes/No)  
l. 
History of peripheral vascular disease in the medical notes (Yes/No)  
m. History of stroke or TIA in the medical notes (Yes/No)  
Index Admission for Myocardial Infarction  
n. Index myocardial infarction (STEMI/NSTEMI)  
o. Coronary artery disease (Single vessel without LMS involvement, Single vessel with LMS 
involvement, Two vessels without LMS involvement, Two vessels with LMS involvement, Three 
vessels without LMS involvement,  Three vessels with LMS involvement)  
p. Percutaneous coronary intervention (Yes/No)  
q. GRACE score (continuous)  

   
 
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Medication at Time of Screening Visit  
N (%) of participants prescribed each of the following categories of medication:   
o 
Aspirin o  
P2Y12 antagonist o  
Statin   
o 
Non-statin lipid lowering therapy  
o 
Beta blocker  o  ACE inhibitor or ARB o  Calcium channel blocker  
o  
Nitrate  o  
Other anti-anginal  
o 
Mineralocorticoid receptor antagonist o  Other diuretic o 
 Anticoagulant  
o 
Insulin  
o 
Oral diabetic medication  
  
5.3  
Primary endpoint  
5.3.1 Endpoint Definition  
Cardiac death or non-fatal recurrent myocardial infarction or unscheduled coronary revascularisation 
between study entry and up to the end of follow-up.  Unscheduled (late) coronary revascularisation is 
defined as revascularisation >6 weeks after screening visit date.  
  
5.3.2 Primary Analysis  
Table: N (%) of participants with primary outcome event by CMA status and overall.    
Table: Numbers of participants whose first event was: a cardiac death, a non-fatal recurrent MI, or an 
unscheduled coronary revascularisation by CMA status and overall.  
Figure: Kaplan-Meier estimates of cumulative incidence from analysis of time from study entry to 
first event of cardiac death or non-fatal recurrent MI or unscheduled coronary revascularisation by 
CMA status with p-value using a log-rank test. This will be the primary statistical analysis.  
Participants who do not have a primary outcome event will be censored at the time of non-cardiac 
death or last follow-up.   
Table: Hazard ratio of primary outcome by CMA status, with 95% CI, and associated p-value using Cox 
regression.  
  
5.3.3 Exploratory analyses of primary outcome  
Relationship between CMA, as a continuous measure of 18F-fluoride uptake, and the risk of suffering 
a primary outcome event will be explored using a Cox proportional hazards regression model with 
(transformed) CMA as the only explanatory variable. A range of potential transformations of CMA will 
be explored, including fractional polynomials, to find an optimal fit to the data.  
A second Cox regression model with (transformed) GRACE score as the only explanatory variable will 
be used to explore the relationship between this risk score and the primary outcome. A range of 
potential transformations of GRACE score will be explored to find an optimal fit to the data. The precise 
version of the GRACE score used, and any necessary details of calculation, will be specified in the 
statistical report.  

   
 
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A third Cox regression model with both GRACE score and CMA as explanatory variables, transformed 
where appropriate, will be used to assess the incremental prognostic value of CMA over and above a 
conventional clinical risk marker.  
Table: Hazard ratio of primary outcome for each explanatory variable, with 95% CI, and associated 
pvalue from each Cox regression model. Also for each model the C-statistic, with 95% CI, as a 
measure of the discriminatory ability.  
  
5.4  
Secondary endpoints  
5.4.1 All-cause death  
Table: N (%) of participants with all cause death by CMA status and overall.  
Figure: Kaplan-Meier estimates of cumulative incidence from analysis of time from study entry to 
death by CMA status with p-value using a log-rank test. Participants who do not have a relevant 
outcome event will be censored at the time of last follow-up.   
Table: Hazard ratio of all cause death by CMA status, with 95% CI, and associated p-value using Cox 
regression.  
  
5.4.2 Cardiac death  
Table: N (%) of participants with cardiac death by CMA status and overall.  
Figure: Kaplan-Meier estimates of cumulative incidence from analysis of time from study entry to 
cardiac death by CMA status with p-value using a log-rank test. Participants who do not have a relevant 
outcome event will be censored at the time of non-cardiac death or last follow-up.   
Table: Hazard ratio of cardiac death by CMA status, with 95% CI, and associated p-value using Cox 
regression.  
  
5.4.3 Non-fatal recurrent myocardial infarction  
Table: N (%) of participants with non-fatal recurrent myocardial infarction by CMA status and overall.  
Figure: Kaplan-Meier estimates of cumulative incidence from analysis of time from study entry to first 
non-fatal recurrent MI by CMA status with p-value using a log-rank test. Participants who do not have 
a relevant outcome event will be censored at the time of death or last follow-up.   
Table: Hazard ratio of non-fatal recurrent myocardial infarction by CMA status, with 95% CI, and 
associated p-value using Cox regression.  
  
5.4.4 Unscheduled coronary revascularisation  
Table: N (%) of participants with unscheduled coronary revascularisation by CMA status and overall.  
Figure: Kaplan-Meier estimates of cumulative incidence from analysis of time from study entry to first 
unscheduled coronary revascularisation by CMA status with p-value using a log-rank test. Participants 
who do not have a relevant outcome event will be censored at the time of death or last follow-up.   
Table: Hazard ratio of unscheduled coronary revascularisation by CMA status, with 95% CI, and 
associated p-value using Cox regression.  
  

   
 
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5.5  
Safety analyses  
5.5.1 To determine the radiation exposure and effective dose associated with 18F-fluoride positron 
emission tomography and computed tomography coronary angiography  
The following will be presented for the scanned population.  Details of radiation exposure for patients 
in the safety population who are not in the scanned population will be put in a footnote.  
Table: mean, standard deviation, median, lower and upper quartile, minimum, and maximum of 
injected 18F-fluoride activity (MBq) and effective radiation dose (mSv) from administration of 
radioactive tracer at baseline visit. The effective radiation dose (mSv) will be calculated from the 
administered dose using the conversion factor of 0.024 mSv/MBq.  Mean, standard deviation, median, 
lower and upper quartile, minimum, and maximum of dose-length product (Gy cm) and effective 
radiation dose (mSv) from computed tomography component of the scans at baseline visit. The 
effective radiation dose (mSv) will be calculated from the dose-length product using the conversion 
factor of 0.014 mSv/Gy cm.  Mean standard deviation, median, lower and upper quartile, minimum, 
and maximum of total effective radiation dose (mSv) at baseline visit.  
The following will be presented for the participants who had a CT scan at 2 years.  
Table: mean, standard deviation, median, lower and upper quartile, minimum, and maximum of 
doselength product (mGy cm) and effective radiation dose (mSv) from the scans at 2-year visit.  The 
effective radiation dose (mSv) will be calculated from the dose-length product using the conversion 
factor of 0.014 mSv/Gy cm.  
These tabulations will not be split by CMA status.  
  
5.5.2. To identify adverse events associated with 18F-fluoride positron emission tomography and 
computed tomography angiography  
The following will be presented for the safety population.  
Table: summary of adverse events within 48 hours of baseline visit showing both numbers and 
percentages of patients with events, and number of events. Also present these numbers split by 
whether events are serious or non-serious, and within seriousness by (i) causality (unrelated or 
possibly related for IMP and NIMP) and MedDRA preferred term (or an alternative classification), and 
(ii) MedDRA system organ class and MedDRA preferred term (or an alternative classification).  No 
formal statistical analysis will be performed.  
Listings with details of each serious adverse event and each non-serious adverse event, separately for 
those within 48 hours of baseline visit and 48 hours of 2-year visit.   
These tabulations and listings will not be split by CMA status.  
  
5.6.  Deviations, Violations  
Listing of protocol violations and listing of protocol deviations.  These will be listed as a separate 
appendix, rather than in the body of the report.  CMA status will be included in the listings.  
  
6.  
Validation and QC  
The following will be done by a second statistician: separate programming and checking of primary 
outcome results and conclusions; the statistical report will be read and sense-checked.  

   
 
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7.  
Data sharing  
A file, or set of files, containing the final data will be prepared, along with a data dictionary.  These will 
be made available to the Chief Investigator at the end of the analysis phase.   
  
8.  
References  
1. SAS® Institute Inc. SAS for Windows. SAS Institute Inc.: Cary, NC, U.S.A