Eicosapentaenoic acid and/or aspirin for preventing colorectal adenomas during colonoscopic surveillance in the NHS Bowel Cancer Screening Programme: the seAFOod RCT

The health burden of colorectal cancer

Colorectal cancer (CRC) continues to represent a huge health burden in the UK. There were approximately 41,300 new cases of CRC (also known as bowel cancer) in the UK in 2014, making it the fourth most common cancer. 2 In the UK, 1 in 14 men and 1 in 19 women will be diagnosed with CRC during their lifetime. CRC is the second most common cause of cancer death in the UK, causing 16,000 deaths in 2014. Worldwide, it has been estimated that nearly 1.4 million cases were diagnosed in 2012. 2 Despite significant advances in the diagnosis and treatment of CRC, overall 5-year survival is currently only 59% (survival figure is for England and Wales for 2010 and 2011). 2

Prevention of colorectal cancer

One strategy to reduce CRC incidence and mortality is prevention. The scientific and clinical rationale for prevention of CRC is well established and is based on the following:

• Knowledge of several environmental and behavioural factors that increase CRC risk, including dietary factors (e.g. red and processed meat intake), excess body weight (i.e. obesity), lack of physical activity, tobacco smoking and excess alcohol consumption. The World Cancer Research Fund has estimated that approximately 45% of CRCs are preventable based on modification of these lifestyle factors. 3

• The long natural history of colorectal carcinogenesis, during which a benign, precursor lesion termed a colorectal adenoma (or polyp) develops and transforms into a malignant neoplasm over a period of years (estimated to be approximately 5–10 years). 4,5

• Improved outcomes for CRC treatment after diagnosis at earlier stages of CRC (98% 1-year overall survival for stage I CRC compared with 40% for stage IV disease; 95% 5-year survival for stage I compared with 7% for stage IV disease). 6,7

Colorectal cancer prevention strategies that are currently used, or are under evaluation, include:

• population screening –

  • early CRC diagnosis by a guaiac faecal occult blood test (FOBt) or a faecal immunochemical test (FIT), conferring secondary benefit from colorectal adenoma identification and removal at colonoscopy

  • detection and removal (by polypectomy) of colorectal adenomas by primary screening endoscopy [colonoscopy or flexible sigmoidoscopy (FS)]

• endoscopic surveillance of high-risk groups, for example individuals with long-standing colitis or previous colorectal adenoma(s)

• chemoprevention (a term first coined in 1976 to describe the use of drugs, vitamins or other nutritional agents to try to reduce the risk of, or delay the development or recurrence of, cancer)

• health education, leading to beneficial lifestyle modification and screening uptake

• promotion of awareness and earlier diagnosis of CRC by education aimed at the public and health-care professionals.

Chemoprevention of colorectal cancer

Despite the undoubted clinical effectiveness of endoscopic polypectomy,8,9 CRC remains a significant problem in screened populations and high-risk surveillance cohorts because of a combination of factors that include suboptimal screening uptake, poor acceptability of endoscopic procedures and ‘interval’ CRC (i.e. those cancers that are diagnosed despite FOBt/FIT and/or endoscopy). 10–13 In a Dutch biennial FIT-based CRC screening programme,14 23% of individuals developed a FIT-interval CRC. The corresponding interval CRC rate for guaiac FOBt programmes is approximately 50%. 15 Moreover, 18 years’ follow-up of the Minnesota guaiac FOBt trial (in which the colonoscopy rate was nearly 40%) found only a 20% reduction in CRC incidence. 13 The UK once-only FS trial10 demonstrated only a 23% reduction in CRC incidence in the intervention group compared with the control group at 10 years. It is also clear that CRC occurs even in patients under close colonoscopic surveillance (1.7 CRCs per 1000 person-years),16 with an estimated post-colonoscopy CRC (PCCRC) rate of between 2% and 9%. 17 Overall, only 10% of CRCs in the UK in 2013 were diagnosed within the UK screening programmes. 18 Therefore, there is still an unmet clinical need for safe and effective primary CRC chemoprevention in combination with existing screening and surveillance programmes.

The natural history and molecular pathogenesis of colorectal carcinogenesis

The molecular pathogenesis of CRC (histopathological term: colorectal adenocarcinoma) has been the subject of several recent reviews. 4,19 In recent years, the original multistage model of cumulative genetic mutations, proceeding through a benign adenoma stage (the so-called adenoma–carcinoma sequence), based on loss of function of the ‘gatekeeper’ tumour suppressor gene adenomatous polyposis coli (APC), which was proposed by Fearon and Vogelstein,20 has been superseded by widespread acceptance that ‘sporadic’ (i.e. not occurring on a background of a distinct genetic predisposition syndrome or inflammatory bowel disease) CRC is not ‘one disease’, but occurs via several pathogenic pathways, which are not mutually exclusive. 21,22

The chromosomal instability (CIN) pathway is characterised by chromosomal abnormalities, including aneuploidy, usually associated with loss-of-function APC mutation and later-stage gain-of-function KRAS mutation. 23 It is exemplified by the rare genetic predisposition syndrome familial adenomatous polyposis (FAP), an autosomal dominant condition that occurs in carriers of a heterozygous germline APC mutation. 24 The microsatellite instability (MSI) pathway is driven by defective deoxyribonucleic acid (DNA) mismatch repair (MMR), leading to accumulation of further somatic mutations (termed MSI-high) including BRAF. 23 It is exemplified by Lynch syndrome, in which carriers of mutations in MMR genes (most commonly MLH1 and PMS2) exhibit increased cancer (including colorectal) risk. 25 Furthermore, a CpG island methylator phenotype (CIMP) pathway is recognised in colorectal adenomas and adenocarcinomas, by which epigenetic changes in DNA methylation lead to altered gene function, in particular silencing of the MMR gene MLH1 leading to defective MMR, which is associated strongly with BRAF mutation. 23 Epigenetic silencing of MLH1 explains the substantial overlap between MSI and CIMP pathways. 4

There are limited data on how early during colorectal carcinogenesis the above phenotypes manifest themselves. 4 There are some data to suggest that CIN features are present in adenomas. 4,26 The CIMP pathway is linked strongly to benign serrated lesions (see below).

Colorectal adenoma

The importance of the benign precursor lesion, which exhibits epithelial cell dysplasia but not invasion of the epithelial basement membrane (termed the adenoma, or adenomatous polyp), as a risk stratification biomarker of future CRC risk but also as a clinically significant lesion (the removal of which is associated with reduced CRC incidence and mortality), has been reviewed in detail. 5

In parallel with more nuanced understanding of the diverse molecular pathogenesis of CRC, the histopathological classification and terminology of the colorectal adenoma has been revised. 5 In particular, hyperplastic-serrated pathway lesions are now acknowledged as separate entities from the more common (conventional) dysplastic adenoma (which can be tubular, villous or mixed tubulo-villous in morphology). Serrated lesions are recognised to have malignant potential per se and may account for 20–30% of CRCs. 27 The World Health Organization’s (WHO’s) WHO Classification of Tumours of the Digestive System28 in 2010 included the term sessile (a term used to recognise that the vast majority of these lesions are ‘flat’ and not polypoid when viewed endoscopically) serrated adenoma. However, the term sessile serrated polyp is now preferred on the basis that the majority of sessile serrated lesions do not display any dysplasia, which is a prerequisite for pathological classification as an adenoma. 5 The traditional serrated adenoma is a separate, rare, fully dysplastic lesion with classical serrated appearances. 27 For the purposes of this report, the term serrated adenoma will continue to be used as the terminology employed continuously throughout the Systematic Evaluation of Aspirin and Fish Oil (seAFOod) trial from 2009 onwards.

Sessile serrated adenomas are more prevalent in the proximal (also known as right) colon (most commonly defined as proximal to the splenic flexure) than the distal (also known as left) colon. 4 Conventional tubular/tubulo-villous adenomas are more uniformly distributed throughout the right and left colon. 4 Molecular features also distinguish between conventional adenomas and serrated adenomas, with a high prevalence of CIN features in conventional adenomas and serrated adenomas commonly displaying a CIMP-high, BRAF mutation-positive, MSI-high phenotype. 4,21,27 Results from a study29 of CRCs suggest that, in reality, there is likely to be a continuous positive gradient of CIMP-high, MSI-high and BRAF mutation frequency in tumours along the distal to proximal colon, rather than an anatomical dichotomy in the distal transverse colon. There is conflicting evidence that a given tumour genotype/phenotype predicts that of synchronous/metachronous lesions, but the majority of data pertain to CRC, not to colorectal adenomas. 4

As direct precursor lesions of CRC, the removal of which is unequivocally associated with decreased future CRC risk,8,9 the colorectal adenoma is a clinically important lesion in its own right. 5 It has been estimated (based on cohort prevalence studies) that approximately 1 in 10–20 colorectal adenomas may eventually acquire a malignant phenotype. 5 The features associated with malignant progression are size, grade of dysplasia and ‘villousness’, namely the degree of villous histological architecture in an individual lesion. 5 On the other hand, the colorectal adenoma can also be considered a biomarker of future CRC risk, regardless of its individual malignant potential. 5 Both colorectal adenoma number and colorectal adenoma size are widely used as the basis for future CRC risk stratification for surveillance after colonoscopy in the UK and elsewhere in the world. 30–33 A common feature of guidelines is the definition of the ‘advanced’ colorectal adenoma based on size (≥ 10 mm), with or without additional histological (e.g. grade of dysplasia, ‘villousness’) features. 30,31,33

Colorectal adenoma measures

Based on widespread acceptance of the number and size of colorectal adenomas as a CRC risk biomarker, the colorectal adenoma has been used as a surrogate colonoscopic end point of reduced CRC risk in chemoprevention trials following polyp clearance at an index procedure (the ‘polyp-prevention trial’). Historically, the presence or absence of any colorectal adenoma [the so-called adenoma detection rate (ADRa)] has been employed as the primary end point in chemoprevention trials, with reliance on this binary end point reflecting the varying quality of colonoscopy between different endoscopists and susceptibility of colorectal adenoma detection to observer variation. 5,34–36 However, this percentage value does not take into account any change in colorectal adenoma number (or size), unlike pre-clinical rodent studies and proof-of-concept clinical studies in FAP patients, in which lesion number and size are routinely measured. 37 More recently, driven by the dramatic improvement in colonoscopy quality and quality assurance (QA) reporting,38 colorectal adenoma number has begun to be reported as a primary outcome in ‘sporadic’ polyp-prevention trials as the mean adenomas per participant (MAP). 39 Population-based studies have consistently demonstrated that colorectal adenoma multiplicity predicts future CRC incidence and mortality. 40–42

Candidate colorectal cancer chemoprevention agents

The existing literature on several potential CRC chemoprevention agents, including non-steroidal anti-inflammatory drugs (NSAIDs), hormone replacement therapy and micronutrients (e.g. folic acid, vitamin D), is well summarised in published reviews. 34,36 The largest body of evidence supports the use of the NSAID aspirin for CRC chemoprevention. 43,44

Anticolorectal cancer activity of eicosapentaenoic acid

Eicosapentaenoic acid is an attractive candidate as a ‘natural’ CRC chemoprevention agent based on several strands of evidence. 60 There is strong pre-clinical evidence that ω-3 PUFAs have anti-CRC activity. 61 However, a systematic review of epidemiological studies has not demonstrated unequivocal benefit from dietary ω-3 PUFA intake on CRC risk. 62 This may be related to the methodological difficulties of measuring ω-3 PUFA or fish intake retrospectively. Alternatively, ω-3 PUFA exposure may not be sufficient for consistent anti-CRC activity in individuals consuming moderate amounts of fish (a portion of oily fish two or three times per week provides only the equivalent of approximately 500 mg per day of EPA and DHA combined). Omega-3 PUFA intake can be increased by ‘over-the-counter’ fish oil supplements, which contain a complex mix of ω-3 and ω-6 PUFAs. 63 However, many of these supplements are associated with a range of minor, troublesome side effects [e.g. eructation (burping), halitosis]. In the prospective VITAL (VITamin And Lifestyle) cohort study, which has uniquely collected data on fish oil supplement use, as well as dietary fish intake, fish oil supplement users were shown to have a 49% reduced CRC risk compared with non-users, an effect primarily observed in men. 64

Purified and concentrated EPA is available in several forms and pharmaceutical formulations. 65,66 EPA alone (without DHA) is available as the free fatty acid (FFA), as a triglyceride (TG) conjugate (the predominant natural form of EPA) or as an ethyl ester (EE) conjugate. 65 Dietary EPA-TG is converted to EPA-FFA in the small intestine by the action of pancreatic lipase, which is released in response to (particularly fatty) food intake. It is unclear which form of EPA is absorbed best from the small intestine and has maximal bioavailability, especially during prolonged use. 65,66 Administration of EPA with food maximises absorption of all forms of EPA. 65 A 500-mg gastroresistant capsule formulation of 99% pure EPA as the FFA has been produced (SLA Pharma AG, Liestal, Switzerland). This formulation was used for the administration of 2 g of EPA-FFA daily as four capsules in the RCT in FAP patients described below. 67 Alternative formulations of purified EPA exist, including a 574-mg formulation of 90% EPA-TG (equivalent to 400 mg of EPA-FFA) in a soft gelatin capsule (Igennus Healthcare Nutrition, Cambridge, UK) that can be used to provide the equivalent 2-g daily dose of EPA-FFA in five capsules.

Eicosapentaenoic acid in all three forms (i.e. FFA, TG and EE) has been demonstrated to have chemopreventive activity in several rodent models of colorectal carcinogenesis, including azoxymethane-induced intestinal tumorigenesis and the Apc^Min/+ mouse model of FAP. 61,68,69 Preliminary evidence that EPA has chemopreventive efficacy in humans was provided by two separate Phase II studies of 2 g of EPA-FFA daily in patients with previous colorectal adenoma(s), which demonstrated a significant reduction in rectal epithelial cell mitosis frequency (not observed with a 1-g daily dose), which was associated with a fivefold increase in rectal mucosal EPA content. 70,71 These studies led to a Phase III RCT of the effect of 2 g of EPA-FFA daily for 6 months on rectal polyposis in patients with FAP (n = 58). 67 This RCT provided the first definitive evidence of chemopreventive efficacy of EPA in humans, with a net decrease in rectal adenoma number and cumulative rectal adenoma size of 22.4% and 29.8%, respectively, between the EPA and placebo groups. 67 The percentage reduction in adenomatous polyp burden was similar to that observed in FAP patients treated with celecoxib,55 a drug that was subsequently demonstrated to prevent ‘sporadic’ colorectal adenomas. 34 In 2012, high dietary intake of marine-derived ω-3 PUFAs was associated with reduced colorectal adenoma risk. 72 The protocol for a RCT of 2.7 g of EPA daily for prevention of rectal aberrant crypt foci was published in 2012 (UMIN000008172), but the trial has yet to report results. 73

Mechanisms of the antineoplastic activity of eicosapentaenoic acid and aspirin

The precise mechanism(s) by which aspirin and EPA have anti-CRC activity is not fully understood. 43,45,61 However, it is currently accepted that, even though these agents are likely to act via multiple COX-dependent and COX-independent mechanisms, modulation of COX activity plays an important role in their antineoplastic effects. EPA and, particularly, aspirin are both potent inhibitors of COX-1, but they alter COX-2 activity in different ways, leading to the production of different bioactive lipid mediators, including PGE₃ (EPA) and 15R-HETE (hydroxyeicosatetraenoic acid) (aspirin). 57 There is some evidence that PGE₃ (unlike pro-tumorigenic PGE₂) has antitumorigenic activity74 and it is known that aspirin-triggered lipoxins derived from 15R-HETE have antiangiogenic properties. 75

Aspirin irreversibly acetylates the COX enzymes. 75 When EPA acts as a substrate for aspirin-acetylated COX-2, it leads to synthesis of 18R-hydroxyeicosapentaenoic acid (18R-HEPE), which can be converted in a 5-lipoxygenase-dependent manner to resolvin (Rv) E1. 75,76 RvE1 has potent anti-inflammatory activity,76 but it is currently not known whether or not RvE1 has direct antineoplastic activity. 77 Specialised pro-resolving (lipid) mediators, such as resolvins and lipoxins, including RvE1, are technically difficult to measure in biological samples and are likely to exert any biological activity at trace concentrations;77 therefore, it remains unclear whether or not sufficient quantities are generated in humans to have meaningful antineoplastic activity. 78

Although RvE1 synthesis provides a hypothesis for a potential interaction between EPA and aspirin, the available clinical evidence suggests that the antiplatelet (COX-1-dependent) effects of EPA and aspirin are simply additive based on the accumulated evidence of extensive use of dual therapy in cardiology patients79 and the effects of the two agents in ex vivo human platelet aggregation studies. 80,81 Colorectal carcinogenesis and atherosclerosis share common pathophysiological mechanisms and clinical risk factors, including obesity. 82 As a consequence, ischaemic heart disease and stroke are common in elderly populations with colorectal neoplasia. 82 Therefore, an attractive feature of CRC chemoprevention using EPA and/or aspirin is the potential for additional vascular benefit in elderly colorectal adenoma ‘formers’ at simultaneous risk of occlusive vascular events. 43,79

A precision-medicine approach to colorectal cancer chemoprevention

A precision or stratified medicine approach to chemoprevention, whereby the need for chemoprevention and the use of a specific agent is determined based on an individual benefit–risk assessment, has yet to be realised.

The preliminary finding of the APACC polyp-prevention trial,83 that the pattern of COX-2 expression in an index colorectal adenoma predicted the preventative efficacy of aspirin, suggests that baseline colorectal adenoma characteristics have potential as predictive biomarkers of individual chemoprevention efficacy.

Red blood cell (RBC) membrane ω-3 PUFA levels (as a validated surrogate biomarker of ω-3 PUFA tissue exposure84,85) have been long established as a biomarker of dietary ω-3 PUFA exposure in cancer epidemiological studies. 86 Between 2014 and 2016, ω-3 PUFA levels were used in RCTs of ω-3 PUFAs as a biomarker of target tissue ω-3 PUFA exposure (termed ‘bioavailability’ here), but also as a possible indicator of compliance and/or placebo group ‘contamination’ by over-the-counter (OTC) ω-3 PUFA use. 67,87,88

In a RCT of EPA-FFA in patients with CRC liver metastasis, tumour EPA content predicted exploratory survival outcomes. 88 However, there was no relationship between the individual rectal mucosal EPA content and the reduction in rectal polyp number in the small RCT of EPA in FAP patients. 67 Therefore, there is a need for further evaluation of RBC and colorectal mucosal ω-3 PUFA levels, as well as novel biomarkers based on the mechanism of action of EPA, as predictors of individual therapeutic response.

There are no validated biomarkers of aspirin anti-CRC activity. However, all the COX-dependent lipid mediators described earlier are measurable by liquid chromatography–mass spectrometry (LC-MS)77,89 and may find utility as therapeutic biomarkers. 77

Safety and tolerability of eicosapentaenoic acid and aspirin

Aspirin and ω-3 PUFAs are already used widely in patient populations, that are relevant to ‘sporadic’ CRC prevention, for prophylaxis following myocardial infarction (aspirin and ω-3 PUFAs), hypertriglyceridaemia (ω-3 PUFAs) and stroke (aspirin). 79

The safety and tolerability of aspirin (≤ 325 mg daily) in previous polyp-prevention trials has been excellent. 34,49 Aspirin use is associated with a dose- and age-dependent increased risk of upper GI and intracranial bleeding. 45,90 Cuzick et al. 53 have put forward the case for a favourable benefit–risk profile for aspirin dosing of ≤ 325 mg daily for 10 years for primary CRC (and other adenocarcinoma) prevention in average-risk individuals aged 50–65 years.

There is little doubt about the safety and tolerability of ‘nutraceutical’ forms of EPA, confirmed by vast experience of intake in healthy human populations. 91–93 Gastroresistant EPA-FFA of 2 g daily has been compared with placebo for up to 6 months in four RCTs, in which tolerability has been excellent. 67,70,71,87 In the RCT involving FAP patients, there was no significant excess of adverse events (AEs) in the EPA-FFA group compared with the placebo group, with only one patient withdrawing from the EPA-FFA group as a result of nausea and epigastric pain. 67 In two Phase II studies of colorectal adenoma patients, there was a slight excess of mild to moderate AEs in the EPA-FFA group compared with the no-treatment70 and placebo groups. 71 In the latter study,71 the GI AEs observed in the EPA-FFA 2-g daily group were not apparent in those taking 1 g of EPA-FFA daily. 71 EPA-TG may be associated with fewer GI AEs, particularly diarrhoea, than EPA-FFA. 66 However, a formal comparison of tolerability between different EPA formulations in a RCT has not yet been undertaken.

Although aspirin and ω-3 PUFAs share antiplatelet activity and both agents prolong bleeding time, excess bleeding episodes with their combined use have not been observed in cardiological practice, in which they are widely used together following myocardial infarction. 79,94 However, clinically significant bleeding events associated with treatment with EPA either alone or in combination with aspirin have, to date, not been monitored in a RCT.

The NHS Bowel Cancer Screening Programme

The NHS Bowel Cancer Screening Programme (BCSP) in England began in 2006. It is currently based on a biennial guaiac FOBt targeted at all individuals aged 60–74 years who are covered by NHS registration data in England (the uptake, based on a returned FOBt kit, is approximately 50–60%). 95 Individuals with an abnormal FOBt (≈2%) are invited for colonoscopy via a specialist screening practitioner (SSPr)-run clinic. All colonoscopy is undertaken by screening-accredited colonoscopists working within a continuous QA framework based on multiple measures, including individual caecal intubation rate, withdrawal time and ADRa. 38,95 Recording of endoscopic findings and subsequent histopathological assessment is also directed by BCSP guidelines and a QA reporting system. 96,97 Any abnormality detected is discussed with the patient at a SSPr follow-up clinic. Detection of a CRC (≈10%, but variable dependent on the number of prevalent vs. incident screening investigations) prompts further management by the local multidisciplinary team (MDT) for CRC. Detection of one or more colorectal adenomas prompts surveillance colonoscopy within the BCSP, as per British Society of Gastroenterology guidelines. 30 Individuals classified as being at ‘low risk’ (i.e. those having one or two subcentimetre colorectal adenomas) are not offered colonoscopy, but remain in the biennial guaiac FOBt programme. Those individuals with three or four small colorectal adenomas (i.e. < 10 mm in size) or one colorectal adenoma of ≥ 10 mm in diameter are classified as being at ‘intermediate risk’ and are offered another colonoscopy at 3 years from the index procedure. Individuals with five or more subcentimetre colorectal adenomas, or three colorectal adenomas with at least one colorectal adenoma of ≥ 10 mm in diameter (i.e. 12% of men and 6.2% of women who undergo screening colonoscopy), are recommended to undergo surveillance colonoscopy 12 months from the screening colonoscopy. 98

Since 2013, the bowel scope programme has been rolled out across England, whereby, in addition to the biennial guaiac FOBt invitation, a single FS is offered to all individuals aged 55 years. 99 The presence of a colorectal adenoma of ≥ 10 mm in diameter, three or more small (i.e. < 10 mm) colorectal adenomas or any adenoma with ‘advanced’ features prompts an invitation for full colonoscopic evaluation, with the combined colorectal adenoma findings from the FS and colonoscopy directing the subsequent surveillance strategy within the BCSP, as described above. 95

The seAFOod polyp-prevention trial

Based on strong proof of concept for primary CRC chemoprevention activity of EPA67 and aspirin,49 the National Institute for Health Research (NIHR)/Medical Research Council (MRC) Efficacy and Mechanism Evaluation (EME) programme funded a 2 × 2 factorial RCT of 2 g of EPA-FFA daily and/or 300 mg of aspirin in ‘high-risk’ individuals identified in the English BCSP. The trial was termed the seAFOod polyp-prevention trial. 100

Primary objective

The primary objective was to determine whether or not EPA prevents colorectal adenomas, either alone or in combination with aspirin. This was addressed by testing the following hypotheses:

• 2 g of EPA-FFA daily is more effective than placebo for reduction in colorectal adenoma recurrence.

• 300 mg of aspirin daily is more effective than placebo for reduction in colorectal adenoma recurrence.

Secondary objectives

The secondary objectives were to assess the tolerability and safety of EPA-FFA and EPA-TG alone and in combination with aspirin.

NHS Bowel Cancer Screening Programme recruiting sites

The English BCSP is organised into local centres of a variable number (usually 1–3) of individual BCSP sites (hospitals undertaking endoscopy), which receive referrals for screening colonoscopy after guaiac FOBt analysis at five regional hubs. Participants identified as ‘high risk’ at participating sites were randomised and followed to surveillance colonoscopy at 12 months. The trial was integrated into the BCSP to utilise routine clinical pathways in order to collect quality-assured data from screening and surveillance colonoscopies.

During the trial, 61 BCSP sites were opened. The date of the first participant, first visit (FPFV) was 11 November 2011; the last participant in was 10 June 2016; and the last participant, last visit (LPLV) was 8 June 2017. Despite approval from the BCSP Research Advisory Committee, widespread engagement from SSPrs and Clinical Research Network (CRN)-funded research nurses (RNs) and a trial extension in 2014, the trial did not recruit to target, recruiting 709 participants against a revised target of 755. The sample size calculation remained at 853, based on the predicted effect size of the interventions, giving 80% power. However, recruitment figures achieved prior to the 2014 extension, the limited recruitment period mandated by the funder and the limitations set by the expiry date on the capsule (EPA-TG) investigational medicinal product (IMP) suggested that recruitment of 755 individuals would be feasible in the extended intervention period (see Statistical methods).

The original strategy was to open 15 recruiting sites from two BCSP hubs (North-East and Eastern). This was based on data from the national BCSP database that suggested that each BCSP centre could identify approximately 50 high-risk patients per year. Trial screening data supported this assumption; however, the eligibility rate was much lower than expected, primarily because of a higher than expected number of screened high-risk individuals who were excluded because of the need for repeat colonoscopy or FS to check for adenoma excision within a 3-month window. Analysis of up-to-date BCSP data in early 2012 revealed that the number of cases requiring repeat endoscopy as part of routine BCSP care had increased nationally during the grant application and set-up phases of the trial. It was determined that a second colonoscopy or FS did not significantly alter the overall ADRa at the 12-month surveillance colonoscopy (see Changes to the protocol). Therefore, the protocol was amended (version 4.0, dated 24 May 2012)100 to include these patients, without loss of statistical power.

A trial site expansion strategy was also implemented in 2012, increasing the number of recruiting sites across England, from Cornwall to Cumbria, to 60 (representing ≈50–60% of English BCSP centres). In late 2015, a decision was made to add one further site that had expressed a strong interest to be involved in the trial.

Delays were experienced in gaining NHS trust research and innovation (R&I) approvals as a result of a general lack of Good Clinical Practice accreditation and research training for BCSP staff, many of whom had not previously contributed to a Clinical Trial of an Investigational Medicinal Product (CTIMP). Sites were supported by the Nottingham Clinical Trials Unit (NCTU) and local CRNs to access training. The median time to gain R&I approval at trial sites was 11.5 (range 4–19) weeks. In addition to a site initiation visit, supplementary training was provided to sites by the co-ordinating centre for participating local investigators, RNs and SSPrs via an instructional video.

In February 2014, recruitment was disrupted significantly when the manufacturer of the original capsule IMP (EPA-FFA) was no longer able to provide stock for the trial. Until an alternative capsule IMP could be identified, approved, manufactured and distributed, sites continued to recruit until local stock was exhausted, at which point that site temporarily suspended recruitment. To maximise recruitment during this period, the top eight most active sites were prioritised for allocation of remaining central stock of capsule IMP. Stock management also ensured that all participants completed the intervention phase of the trial using the same EPA formulation (FFA or TG). This strategy enabled the trial to continue recruiting between February and October 2014, after which a new capsule IMP became available. Partly because of this delay, a 36-month extension was approved (in October 2014) by the NIHR EME board in order to complete trial recruitment.

Baseline visit

Individuals attending a routine outpatient follow-up BCSP appointment to obtain results of the screening colonoscopy were approached. Individuals who were eligible and willing to take part in the trial were asked to provide written informed consent. Demographic information and details of participants’ medical histories were collected and participants were randomised. It was preferred that participants were randomised within 4 weeks of the first complete BCSP screening colonoscopy. However, to maximise recruitment, randomisation was allowed outside this time window as long as it was recorded on the protocol deviation log. A prescription was issued for the supply of IMP for 6 months. The local hospital pharmacy dispensed the trial treatment. A second prescription for a further 6 months of IMP was provided at visit 4.

Biological samples were taken, comprising a blood sample [2 × 6-ml K₂EDTA (ethylenediaminetetraacetic acid) Vacutainer^® tubes; Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA] and a urine specimen of 5–10 ml. Samples were taken only if the participant provided separate, specific consent for collection of blood and urine.

In addition, participants were asked to complete a pre-treatment Food Frequency Questionnaire (FFQ) so that any change in dietary ω-3 PUFA intake during trial involvement could be determined.

Visits 2 and 3: telephone calls at 2 and 12 weeks

Participants were contacted by the SSPr/RN by telephone at 2 and 12 weeks after starting trial treatment. Participants were asked about any symptoms or new medical problems since the last contact and were reminded to take the IMP as directed.

Participants who were due to undergo a repeat colorectal endoscopic procedure between visits 2 and 3 were reminded to discontinue IMP temporarily. Colonoscopic findings at the repeat procedure were collected and recorded in the same way as for the baseline visit.

Visit 4: outpatient visit

At 6 months, participants were invited to attend the BCSP site, at which time mid-treatment blood and urine specimens were collected from those who had provided consent. Participants were asked about any symptoms or new medical problems since the last contact. Any unused trial treatment from the first prescription was collected and counted. Each participant then received a new prescription for trial treatment for a further 6 months.

Visit 5: telephone call

A third telephone contact was conducted with participants at 38 weeks after starting the trial treatment. The SSPr/RN asked about any symptoms or new medical problems since the last contact and reminded the participant to take trial treatment as directed.

Visit 5a: telephone call

An extra telephone contact was conducted for participants who had a repeat full colonoscopy between visits 2 and 3 after starting the trial treatment. The SSPr/RN asked about any symptoms or new medical problems since the last contact and reminded the participant to take his/her trial treatment as directed. The SSPr/RN liaised with the hospital pharmacy about delivery of a third dispensing of IMP to these participants. The relevant participants then received a new prescription for trial treatment for a further 3 months.

Visit 6: surveillance colonoscopy

Participants attended for routine surveillance colonoscopy at 12 months from the date of the screening colonoscopy. Participants took the final dose of trial treatment on the day before surveillance colonoscopy. Blood and urine specimens were obtained, as well as four random biopsies of macroscopically normal rectal mucosa (at least 2 cm from any polyp) at the end of the surveillance colonoscopy.

Colorectal adenoma outcomes at the 12-month surveillance colonoscopy were collected as per usual BCSP practice, including the number, size (maximum dimension in mm from the histopathology report, or the endoscopic size if the adenoma was not retrieved or was removed by hot biopsy), site [proximal to the splenic flexure (right) or at/distal to the splenic flexure (left)], histological type (tubular/tubulo-villous, villous, serrated) and presence of high-grade dysplasia of all colorectal adenomas.

Visit 7: routine post-colonoscopy visit

Participants were seen after surveillance colonoscopy as part of routine BCSP follow-up, during which a second FFQ was completed. Participants had the option to complete the FFQ at visit 6 or over the telephone if they decided to receive colonoscopy results by telephone.

Randomisation

Participants were registered in the trial using a secure web-based randomisation system. Randomisation was based on a computer-generated, internet-based treatment assignment determined by a pseudo-random code using random permuted blocks of randomly varying size, created by NCTU. Participants in the trial were allocated with equal probability to either treatment group. It was planned to stratify by BCSP centre. However, after database lock, it was discovered that BCSP site had been used, rather than BCSP centre. As sites could be associated only with an individual BCSP centre, this still ensured balance between centres.

Participants were randomised to a simple 2 × 2 factorial design (Table 1) to:

• 2 g of EPA-FFA, or an equivalent FFA dose of 90% EPA-TG (2780 mg), daily by mouth, or their identical placebos (capric and capryllic acid medium-chain TGs for both formulations)

in addition to:

• 300 mg of enteric-coated aspirin daily by mouth (as one 300-mg tablet taken with food) or identical placebo.

The sequence of treatment allocations was concealed until interventions had all been assigned and recruitment, data collection and all other trial-related assessments were completed. The actual allocation was not divulged to either the staff at the BCSP site or the participant. The trial prescription produced by the randomisation system referenced specific trial treatment containers. The trial drug prescription was signed by the local PI or a co-investigator, as defined by the site delegation log.

Interventions

The IMPs used in this trial were gastroresistant capsules of 99% pure EPA in the FFA form (EPA-FFA), 90% EPA as the TG conjugate (EPA-TG) in soft gelatin capsules, enteric-coated aspirin tablets and their identical placebos.

In February 2014, the supplier of the EPA-FFA IMP (SLA Pharma AG) disclosed that it could no longer provide further capsule IMP to the trial. The Trial Management Group (TMG), NIHR EME programme and the Trial Steering Committee (TSC) made the decision to continue the trial using an alternative EPA formulation and identical (medium-chain TG) placebo. Close consultation with the Medicines and Healthcare products Regulatory Agency (MHRA) and the Research Ethics Committee (REC) was also undertaken. A substantial amendment (number 14) was approved by the REC on 26 August 2014 and the trial received a Clinical Trials Authorisation (CTA) from the MHRA on 29 August 2014 for a new capsule IMP (EPA-TG). The new formulation maintained FFA equivalence (2000 mg daily) with the previous EPA-FFA formulation by using 5 × 574-mg 90% EPA-TG capsules per day (a total of 2870 mg), taking into account the percentage weight per weight (w/w) content of EPA and the presence of the glycerol backbone in the re-esterified TG.

The CTA required a specific simplified IMP dossier for the 90% EPA-TG, detailing its multistep manufacture, and also a cover document for the existing investigator brochure, which compared the chemical structure, GI absorption, bioavailability and tolerability profiles of the FFA and TG forms of EPA.

Although the 90% EPA-TG capsules that were proposed as a new IMP were publicly available for purchase as a nutritional supplement (from Igennus Healthcare Nutrition), the MHRA requested a programme of stability testing to meet manufacturing QA requirements for a CTIMP (see Appendix 1). A programme of accelerated (30 °C, 65% relative humidity) and standard (25 °C, 60% relative humidity) stability testing of capsules began in July 2014 (performed by ALS Food & Pharmaceutical, Carlisle, UK) and was performed once every 3 months. The core data set comprised the peroxide value (POV), para-anisidine value (pAV) and derived total oxidation (TOTOX) value, as well as a full PUFA analysis, to determine the EPA content. Data from initial accelerated testing at 3 months gave a minimum 12-month shelf life under standard conditions for the trial to continue with the new capsule IMP. Rolling stability testing provided a continuous extension of shelf life until 12 June 2016, when capsule IMP use ceased, as per the maximum approved shelf life (3 years) of the capsule IMP approved by MHRA.

The commercially available 90% EPA-TG capsule was also encapsulated with a differently coloured soft gelatin coat (olive green) to be able to produce an identical placebo because of the difference in appearance of EPA and medium-chain TG oils.

Participants took only one formulation of the EPA, which was either FFA or TG, or its matching placebo. Stocks of the IMP were managed during the transition period from the FFA formulation to the TG formulation to ensure that sites had sufficient stock of EPA-FFA and placebo capsules to manage existing and new participants throughout each individual intervention period.

The trial treatment was taken daily from the date of randomisation to the day before the 12-month surveillance colonoscopy.

Blinding

Participants, SSPr/RNs, local investigators and those assessing the outcomes were all blinded to treatment allocation. The statistical analysis for the trial was also blinded until data were locked, except for independent Data Monitoring Committee (DMC) reports.

Unblinding

Access to the sequence of treatment allocations was confined to the NCTU data manager and a central pharmacy, in case of out-of-hours unblinding. In the event of the need to break the code, the date and reason were recorded on the web-based unblinding system. The local hospital pharmacy had access to the web-based unblinding system in normal office hours and out-of-hours access was provided via the sponsor at St James’s University Hospital. The requirement for unblinding was considered low. All participants were given a trial identification card, containing details of the IMPs, which participants were encouraged to show when seeking advice or management from any health professional. Unblinding did not occur during the trial.

End of the trial

Participants left the trial when they completed their routine post-surveillance colonoscopy visit (visit 7).

Cases of failure to receive allocated treatment and withdrawal from follow-up were reported, and the reason(s) for withdrawal (if given) were documented. If a participant did not receive allocated treatment but agreed to remain in the trial, outcome data collection continued in accordance with the protocol. Participants were informed at the start of the trial that data collected up to the point of withdrawal would be retained and used in the final analysis.

Trial withdrawal

Participants could withdraw from the intervention or the trial at any time without giving a reason and without compromising future management. Data collected up to the point of withdrawal were retained for the purposes of the intention-to-treat (ITT) analysis. Participants could withdraw from the intervention only but continue in the trial, thereby completing outcome measures. To maximise primary outcome data collection, BCSP surveillance colonoscopy data were also collected from participants who withdrew from the trial, as per the informed consent.

Primary outcome

The primary outcome was the number of participants with one or more colorectal adenomas detected at the first BCSP surveillance colonoscopy 12 months after the screening examination (ADRa).

Secondary outcomes

The secondary outcomes were as follows:

• Total number of colorectal adenomas per participant at BCSP surveillance colonoscopy (total MAP).

• Detection of one or more ‘advanced’ (i.e. ≥ 10 mm in diameter, high-grade dysplasia or villous histology) colorectal adenomas at the 12-month BCSP surveillance colonoscopy (advanced ADRa).

• Number of ‘advanced’ colorectal adenomas per participant at the 12-month BCSP surveillance colonoscopy (advanced MAP).

• Detection of one or more conventional adenomas (conventional adenoma end points were defined after database lock) at the first BCSP surveillance colonoscopy (conventional ADRa).

• Number of conventional adenomas (conventional adenoma end points were defined after database lock) per participant at the first BCSP surveillance colonoscopy (conventional MAP).

• Detection of one or more serrated adenomas at the first BCSP surveillance colonoscopy (serrated ADRa).

• Number of serrated adenomas per participant at the first BCSP surveillance colonoscopy (serrated MAP).

• The region of the colorectum (right colon: any part of the colon proximal to the splenic flexure; left colon: the rectum and the colon at/distal to the splenic flexure) in which adenomas are detected at the first BCSP surveillance colonoscopy.

• Reclassification from ‘high risk’ to ‘intermediate risk’ after the first BCSP surveillance colonoscopy (BCSP risk stratification at the first surveillance colonoscopy states that any individual who does not continue to fulfil ‘high-risk’ criteria is classified as ‘intermediate risk’ for further colonoscopic surveillance at 3 years).

• Detection of CRC prior to, or at, the first BCSP surveillance colonoscopy.

• Dietary fish and other seafood intake at baseline and at the end of the trial.

• Red blood cell EPA and rectal EPA levels at baseline, 6 months (RBC only) and 12 months from randomisation.

• Absolute RBC fatty acid (i.e. DHA, AA, EPA-to-AA ratio) levels and difference from baseline at 6 months and 12 months.

• Rectal mucosal fatty acid (i.e. DHA, AA, EPA-to-AA ratio) levels at surveillance colonoscopy.

• Adverse events, including clinically significant bleeding episodes (i.e. haemorrhagic stroke or GI bleeding requiring hospital admission or investigation).

Protocol deviations

The protocol defined a protocol violation as:

• > 50% of trial medication returned in total

• inadvertent use of OTC medication containing aspirin, NSAIDs or fish oil for > 2 weeks in total

• surveillance colonoscopy occurring outside the allowed time windows (48–52 weeks after the last complete screening BCSP colonoscopy, or 60–64 weeks for participants undergoing a repeat full colonoscopy within 3 months of initial screening).

The protocol deviation log collected the above violations and any additional protocol deviations (i.e. any deviation from the protocol that occurred during a participant’s time in the trial, whether deliberate or non-deliberate).

Trial oversight

Oversight committees were assembled to ensure the proper management and conduct of the trial, and to uphold the safety and well-being of participants. The general purpose, responsibilities and structures of the committees were described in the protocol.

Risk assessment and safety monitoring

A risk assessment was conducted as part of protocol development and was monitored regularly throughout the trial for new risks. The main risks to the trial were reliance on the BCSP to recruit to a large multicentre RCT, as the BCSP had not previously hosted CTIMP research and staff were not experienced in recruiting participants. Compliance with trial medication and drug accountability was reliant on participants returning medication and patient reports, as well as staff maintaining detailed records of medication logs.

Recruitment sites were supported by the NCTU, which initially provided training through a site initiation visit, as well as supplementary training, as required. Investigators had access to a research area of the trial website (www.seafood-trial.co.uk) and access to the NCTU trial team for day-to-day queries. To obtain high-quality data, regular central monitoring checks were performed according to the monitoring plan, including review of recruitment, retention and data collection rates by the TMG, TSC and DMC.

Data on AEs and serious adverse events (SAEs) were collected. As agreed by the sponsor, REC, DMC and TSC, the DMC was provided with a listing of all AEs and SAEs, including any deaths (if applicable), at each DMC meeting.

As part of the switch from the EPA-FFA formulation to the EPA-TG formulation, a review was undertaken of the Reference Safety Information (RSI) for the trial. Evidence was provided in a supplement, including MHRA-stipulated RSI for the EPA formulations, version 2.0, dated 22 August 2016 (approved by the MHRA on 3 October 2016) to the investigator brochure version 6, dated 10 January 2014, that the FFA and TG formulations were likely to have a similar pharmacological and risk/AE profile, as well as similar anti-CRC efficacy, in the seAFOod trial population [see the project web page: www.journalslibrary.nihr.ac.uk/programmes/eme/0910025/#/ (accessed 25 April 2019)].

Monitoring

Following an internal assessment of the trial by the quality assurance manager at the NCTU, the trial was assessed as a medium-risk trial requiring low-intensity monitoring. Based on this risk category and the specific risks identified, the monitoring strategy consisted of on-site routine and triggered monitoring visits, central monitoring and review of regular reports by trial oversight committees, as well as assessment and oversight of third-party vendors. On-site visits were conducted by either the trial manager or a trial monitor. Central monitoring was conducted by the NCTU and reviewed regularly by the TMG, the TSC and the DMC. On-site and central monitoring revealed no major common concerns during the trial. Sites that received an on-site monitoring visit at some point during the trial accounted for 68% of randomised participants.

Patient and public involvement

There was PPI throughout the trial. One PPI member was integral to trial design, grant application and production of participant-facing trial materials. He then took no further part in the trial. Subsequent PPI input on the TSC was provided by a different PPI member. Both PPI representatives brought personal experience of the BCSP pathway to their roles. Two PPI representatives from Independent Cancer Patients’ Voice contributed to the production of site and participant results summaries and to the development of the dissemination plan for the trial findings.

Payments to participants

Participants were not paid to participate in the trial. Visits 1, 6 and 7 were planned to co-ordinate with routine BCSP appointments. However, an additional visit at 6 months (visit 4) was required; therefore, participants were offered a reimbursement of travel expenses up to a maximum of £10.

The seAFOod polyp-prevention trial biobank

Blood, urine and rectal mucosa sample collection in the seAFOod polyp-prevention trial is described in detail in an appendix of the trial protocol, which has been published. 100

In brief, a blood sample and a urine specimen were obtained at visits 1, 4 and 6. Blood was immediately separated into plasma, leucocytes and RBCs. Rectal biopsies were obtained only at the surveillance colonoscopy at visit 6.

Sample cryovials were stored in either a Liebherr Underbench –20 °C freezer (Liebherr Group, Bulle, Switzerland) supplied by the seAFOod trial or the existing trust freezer facility at –20 °C or colder, in PathoSeal bags (DGP Intelsius Ltd, York, UK). Storage temperature was monitored using a Hanna HI-141 CH Datalogger (Hanna Instruments Ltd, Leighton Buzzard, UK), or a similar system provided by the trust site, and recorded on sample worksheets.

University of Bradford staff, in close collaboration with the NCTU, organised the collection of samples from each BCSP site approximately every 6 months by a specialist courier (CitySprint Health, London, UK). The original proposal was to transfer samples to the central biobank facility every 3 months. However, the expansion of participating trial sites in 2012–13 meant that the collection strategy was revised. BCSP sites were grouped into 10 individual routes by CitySprint Health so that collection from these sites could be scheduled for the same day. Sites were asked to confirm whether or not they required a collection, and the number of PathoSeal bags at that site, to allow CitySprint Health to supply the correct thermal box size. If sites were unable to accommodate a collection on their scheduled route day, individual collections were arranged. The day before collection, CitySprint Health arranged for pre-addressed thermal boxes, which contained dry ice, to be sent to appropriate BCSP sites. The lid of the thermal box was removed only when necessary for adding samples. All sealed PathoSeal bags collected since the last courier sample collection were placed in the thermal box and dry ice was spread around the bags to ensure that they were completely covered. The thermal boxes were delivered to the University of Bradford on the same day or, in the case of routes involving longer distances, the following morning. No units were received with low dry ice levels and all samples were received frozen.

Each BSCP site completed a shipment form that documented the samples being transported. A carbon copy of the shipment form was kept in the site file with the carbon copy of the completed sample worksheets. The original shipment form and the original completed sample worksheets were sent with the samples.

On receipt of each shipment of transportation units at Bradford, a sample tracking form was completed. Contents were then checked against the sample worksheet. Cryovials were transferred into sample boxes (10 × 10 cell cryoboxes, manufactured by Sarstedt, Nümbrecht, Germany) specific to each sample type, and cryoboxes were kept on ice at all times when removed from the freezer. All details were entered onto a separate sample tracking form for each thermal box. Samples were stored in type-specific cryoboxes and racks, minimising disruption when samples were removed for analysis.

All samples were stored in a dedicated –80 °C freezer (New Brunswick™ U570 –80 °C; Eppendorf, Hamburg, Germany), which was connected to the emergency power supply at the Institute of Cancer Therapeutics, University of Bradford, and supported with a CO₂ back-up system (New Brunswick) and Centroller AD11+ (Centroller, Staines, UK) telephone alarm system. Freezer temperature was monitored daily.

The details from the sample tracking form and the sample worksheets were entered into the seAFOod trial biobank sample database. The database allocated a unique Clinical Trials Pharmacology Laboratory (CTPL) tracking identification (ID) to each participant-specific set of samples in a PathoSeal bag (i.e. if a participant had samples taken on three visits, they received three CTPL IDs). The CTPL ID was marked on the sample worksheet and the sample tracking form. The database was stored on the University of Bradford server, in the ‘Secure’ section, accessible only by nominated CTPL staff, and backed up regularly, as described in the University of Bradford’s computer policy. The database was the primary source for sample tracking; however, paper documents were available as a secondary source if necessary, stored in participant and shipment folders in the secure Human Tissue Act-approved laboratory. 107

Samples were obtained at BSCP sites between FPFV (i.e. 11 November 2011) and LPLV (i.e. 8 June 2017). The first samples received at Bradford were delivered on 18 July 2012 and the last sample shipment took place on 27 July 2017. In the intervening time, 332 collections were made from BCSP sites, with 1775 individual PathoSeal bags received.

Samples were stored at BSCP sites for between 1 and 696 days (mean 124 ± 92 days, median 115 days). There were 1378 (78%) sample sets stored at BSCP sites for < 6 months. Thirty (2%) sample sets were stored at BSCP sites for > 12 months.

The majority of sample sets (n = 1021; 58%) were stored in BCSP sites at –20 °C (range –16 to –24 °C), with 230 (13%) sample sets stored at –40 °C (range –25 to –69 °C) and 524 (30%) sets stored at or below –70 °C.

One or more biological samples were received from 677 of 709 (95%) randomised seAFOod trial participants. Of the 709 participants, 73% (519) of participants provided full sample sets of blood, urine and rectal mucosa from all three visits. There were 76 participants who provided samples at two visits (visits 1 and 4, n = 49; visits 4 and 6, n = 15; visits 1 and 6, n = 12). Eighty-two participants provided samples at only a single visit (visit 1, n = 75; visit 4, n = 2; visit 6, n = 5).

Overall, a total of 7322 biological samples were received (16,258 sample aliquots):

• 1715 plasma samples (6746 aliquots)

• 1714 leucocyte samples (1714 aliquots)

• 1707 RBC samples (3421 aliquots)

• 1664 urine samples (3309 aliquots)

• 522 rectal biopsies (1068 aliquots).

Compliance with biological sample collection [defined as the proportion of sample sets expected (n = 2127) that were received with at least one sample aliquot] was 80% (blood), 78% (urine) and 74% (rectal mucosa).

Laboratory protocol deviations were monitored carefully by sample worksheets:

• 80% of blood samples were obtained per protocol (centrifugation within 30 minutes and transfer to the freezer within 60 minutes).

• 2% of sample sets were received without worksheets.

• 1% of samples sets were received with insufficient data to assess timing.

• 8% of blood was separated after 30 minutes, but frozen within 1 hour of collection.

• 9% of blood samples took > 1 hour to separate (often related to split hospital sites for endoscopy and sample handling).

A small number of other protocol deviations were noted, including:

• 1% of sample sets that suffered a temperature deviation (but no thaw).

• 1% of sample sets that defrosted at some point.

• 0.5% of sample sets suffered ‘other’ deviations (including wrong anticoagulant blood tube used and biopsies placed in formalin).

Fatty acid measurement and analysis

The methods used are described in detail by Volpato et al. 89 In brief, fatty acids were extracted from washed RBC membranes, or rectal mucosal homogenates, using an isopropanol/chloroform method with acid hydrolysis, in order to measure the total membrane/tissue fatty acid pool. 89 Liquid chromatography, in combination with electrospray ionisation triple quadrupole tandem mass spectrometry (ESI-MS), was performed with a Waters Alliance™ 2695 High Pressure LC module (Waters, Milford, MA, USA) in combination with a Waters Micromass™ Quattro Ultima triple quadrupole mass spectrometer on derivatised samples, in the presence of internal standard (deuterated ALA), as described by Volpato et al. 89 Data are expressed as the percentage of each fatty acid relative to the total fatty acid peak chromatographic area for the ω-3 PUFAs C18:3 (ALA), C20:5 EPA, C22:5 docosapentaenoic acid (DPA) and C22:6 DHA; the ω-6 PUFAs C18:2 linoleic acid (LA) and C20:4 AA; and the monounsaturated C18:1ω-9 oleic acid and saturated fatty acids C18:0 stearic acid and C16:0 palmitic acid. 89

Measurement of dietary fish intake

Participants were asked to complete a FFQ at baseline (visit 1) and at the end of the intervention (visit 6 or 7). The validated European Prospective Investigation of Cancer (EPIC) short FFQ was used, which is a semiquantitative, paper-based FFQ that includes 130 food items and supplementary questions about use of fat, milk, cooking methods, salt and supplement use. 108 The FFQ measures an individual’s habitual food intake over the preceding 12 months. The primary purpose of the FFQ was to determine if there was any change in dietary marine ω-3 PUFA intake during trial participation. As fish is the primary source of bioactive ω-3 PUFAs EPA and DHA, the reported consumption of fish was analysed before and after intervention with IMP.

Total fish consumption and consumption of oily fish were considered separately. Six FFQ food item variables were used for the analysis of total fish (i.e. fried fish, fish fingers, white fish, oily fish, shellfish, roe). Consumption of oily fish was based on a single food item variable (oily fish). Participants indicated the frequency of food consumption, ranging from ‘never or less than once per month’ to ‘six times per day’. This was recoded into frequency per day (Table 2). The consumption of total fish per day was calculated by summing the reported fish consumption for each of the six fish variables.

The data were used to recode participants into four categories (i.e. never, low, middle or high), based on their weekly consumption of total fish and oily fish (Table 3). Categorisation of weekly fish consumption was based on the Scientific Advisory Committee on Nutrition recommendation to consume two portions of fish per week, one of which should be oily. 109

Further analysis of the dietary PUFA intake data is planned and will include full nutrient and food group analysis using FETA (FFQ EPIC Tool for Analysis) software (www.srl.cam.ac.uk/epic/epicffq/; accessed April 2018).

Planned laboratory studies

The nested laboratory studies originally planned for the EME-funded seAFOod trial project are described in the trial protocol version 6.0, dated 11 August 2014 [see the project web page: www.journalslibrary.nihr.ac.uk/programmes/eme/0910025/#/ (accessed 25 April 2019)]. The switch in capsule IMP that occurred in 2014 required a more detailed PUFA analysis than was originally planned to test equivalence of the bioavailability of FFA and TG formulations measured by RBC and rectal mucosal EPA content. Therefore, funding for much of the planned biomarker work was diverted to a more extensive PUFA analysis.

Changes from the protocol to the statistical analysis plan

A number of changes to details contained within the protocol are documented in the SAP (version 1.1, dated 24 August 2017) [see the project web page: www.journalslibrary.nihr.ac.uk/programmes/eme/0910025/#/ (accessed 25 April 2019)]. These are listed in Table 4.

In addition, the changes documented in Table 5 were made after database lock and release of the treatment codes.

Sample size

The original sample size estimate of 904 participants (to ensure 768 evaluable patients and assuming a 15% loss to follow-up) in trial protocol versions 1–3 was based on a RCT of the same dose and preparation of EPA-FFA in FAP patients,67 a meta-analysis of aspirin RCTs49 and detailed 2007–8 audit data from the South of Tyne and Tees BCSP centres.

For the protocol revision in May 2012 (related to inclusion of patients who required a repeat endoscopic procedure during the 3-month screening window), the sample size was re-estimated using audit data on surveillance colonoscopy in 1189 patients classified as ‘high risk’ in 2010, from the North-East BCSP hub (nine BCSP centres) and the Southern BCSP hub (17 BCSP centres). A total of 930 (78%) patients went straight to surveillance after a single screening colonoscopy. Colorectal ADRa data at surveillance colonoscopy were available for 738 patients and, of these, one or more colorectal adenomas were detected in 465 (63%) patients. Corresponding figures for ‘high-risk’ patients having a repeat partial colonoscopy or FS within 3 months of an initial screening colonoscopy showed that one or more adenomas were detected at first surveillance colonoscopy in 59 out of 110 (54%) patients, and for patients having a repeat full colonoscopy, one or more adenomas were detected in 56 out of 83 (67%). The overall ADRa at first surveillance colonoscopy was 62%, which was consistent with the original estimate of 60%; therefore, the sample size remained unchanged.

To detect a minimum 18% relative reduction in adenoma risk in each two-group comparison [less than the 22% reduction in polyp number compared with placebo in the FAP trial67 and below the absolute reduction in polyp number at 1 year (38%) in aspirin RCTs49] from a 60% adenoma recurrence rate at surveillance colonoscopy to 49%, 678 evaluable ‘high-risk’ individuals were required to be randomised equally to the four treatment groups, with 80% power at a 5% two-sided significance level.

Standard practice for 2 × 2 factorial designs, in the absence of an interaction, bases the sample size estimate on the two-group comparison of treatment versus placebo (and divides the total equally between the four groups). With the sample size of 678 based on this method, there is, in fact, a slight reduction in power (to 75%), which arises if both treatments work, because then the overall comparison for treatment A is not 0.49 versus 0.6, but is 0.445 versus 0.545 (averaging over the placebo and treatment B groups). To keep power at 80% for the above figures, a simulation using Stata^® version 10 (StataCorp LP, College Station, TX, USA) and employing the proposed analysis method indicated that 192 individuals were required per group (a total of 768 evaluable ‘high-risk’ individuals).

In trial protocol versions 1–3, a 15% drop-out rate was assumed. However, feedback from BCSP sites and experience from the first few months of the trial suggested that the drop-out rate of ‘high-risk’ BCSP patients was < 15%. Allowing for a 10% drop-out rate, the proposed sample size increased to 768/0.9 = 853 individuals.

For the purposes of the trial extension granted by the EME board in 2014, we proposed a revised realistic recruitment target of 755, which provided 71% power to detect the same effect size as above, still assuming a 10% drop-out rate.

Analysis plan

A SAP was finalised prior to database lock and release of treatment codes to the statistician. All summaries and statistical analyses were conducted using Stata version 15.0.

Analysis populations

• Intention-to-treat population: all randomised participants with post-randomisation data. Analysis was according to the treatment group to which they were randomised.

• Safety population: all randomised participants who had at least one dose of trial medication. Analysis was according to the treatment they actually received.

• Per-protocol population: all randomised participants not deemed to have a major protocol violation. Major protocol violations that resulted in exclusion from the per-protocol population were –

  • had not taken sufficient medication (participants who had taken ≤ 75% of their expected EPA and/or ≤ 50% of their expected aspirin)

  • found to be ineligible post-randomisation

  • any use of OTC medication containing aspirin, NSAIDs or fish oil for > 2 weeks during the treatment period.

The final composition of the per-protocol population was determined by final data review prior to the treatment codes being revealed.

Analysis of the primary end point was based on the ITT population. The analysis was repeated based on the per-protocol population. This was considered supportive to the primary analysis.

Analyses of all secondary end points, with the exception of AEs, were based on the ITT population.

Summaries of the AEs were based on the safety population.

Missing data

Sensitivity analyses were performed to support the primary analyses. All secondary analyses assumed that the data were missing at random and no imputation was performed.

Primary outcome

The primary outcome was the number of participants with one or more colorectal adenomas detected at the first BCSP surveillance colonoscopy 12 months after the screening examination (the ADRa).

The primary outcome was analysed by an ‘at-the-margins’ approach,110 after first examining whether or not there was any evidence of an interaction between EPA and aspirin. An ‘at-the-margins’ approach analyses all participants randomised to EPA (i.e. EPA + aspirin plus EPA + placebo aspirin, combined) versus all participants not randomised to EPA (i.e. placebo EPA + aspirin plus placebo EPA + placebo aspirin, combined), and all participants randomised to aspirin versus all participants not randomised to aspirin. Given that there was no evidence of an interaction, the log relative risk was estimated using a mixed-effects log-binomial regression model, with BCSP site included as a random effect, and the risk differences and ratios presented. Both interventions were fitted simultaneously and the analysis was adjusted for ‘repeat colorectal endoscopic procedure within 3 months required’ and BCSP site. This was a change to the analyses defined in the SAP, which stated that BCSP centre would be included as a random effect.

Sensitivity analyses for the primary outcome

The following sensitivity analyses were performed on the primary end point:

• Analysis using the per-protocol population.

• Analysis as a multilevel model. Some BCSP centres comprise multiple hospital sites; therefore, a sensitivity analysis was conducted in which both BCSP centre and site were treated as random effects in a multilevel model.

• Multiple imputation of missing primary end-point data.

• Further adjustment of baseline variables with any marked imbalance, if appropriate.

• Investigation of the effect of treatment adherence using complier-average causal effect (CACE) estimation methods. 111,112 ITT analysis does not represent the treatment effect of non-compliance with treatment; therefore, CACE analysis was deemed to be important if any treatment effect was directly affected by the level of compliance. The percentage of the required total dose taken by participant from randomisation to first surveillance colonoscopy (both in binary and continuous form) was included in the model as an instrumental variable to estimate such an effect.

• Further adjustment for EPA capsule formulation, that is EPA-FFA (or placebo) or EPA-TG (or placebo).

• Further adjustment for oily fish intake.

• Further adjustment for baseline RBC EPA levels.

Secondary outcomes

• The total number of colorectal adenomas per participant at the 12-month BCSP surveillance colonoscopy was analysed using a Poisson regression model. The incidence rate ratio (IRR) and 95% CIs were presented.

• The number of participants with an ‘advanced’ colorectal adenoma (≥ 10 mm maximum dimension, high-grade dysplasia or villous histology) at the first BCSP surveillance colonoscopy (advanced ADRa) was analysed using a log-binomial regression model. The risk difference and 95% CIs were presented.

• The number of ‘advanced’ colorectal adenomas per participant at the first BCSP surveillance colonoscopy (advanced MAP) was analysed using a Poisson regression model. The IRR and 95% CIs were presented.

• The number of participants with one or more conventional colorectal adenomas at surveillance colonoscopy (conventional ADRa) was derived from the total and serrated adenoma data and was analysed using a log-binomial regression model. Risk difference and 95% CIs were presented.

• The number of conventional colorectal adenomas per participant at surveillance colonoscopy (conventional MAP) was derived from the total and serrated adenoma data and was analysed using a Poisson regression model. The IRR and 95% CIs were presented.

• The number of participants with one or more serrated adenomas at surveillance colonoscopy (serrated ADRa) was analysed using a log-binomial regression model. The risk difference and 95% CIs were presented.

• The number of serrated adenomas per participant at surveillance colonoscopy (serrated MAP) was analysed using a Poisson regression model. The IRR and 95% CIs were presented.

• The region of the colorectum (right colon: any part of the colon proximal to the splenic flexure; left colon: the rectum and the colon at/distal to the splenic flexure) that colorectal adenomas were detected at the first BCSP surveillance colonoscopy was explored, using a Poisson random-effects model with bivariate response (corresponding to adenoma counts in the left and right colon), in which treatment and a baseline adenoma count were independent variables together with random intercepts corresponding to participant and BCSP site. The IRR and 95% CIs were presented.

• The number of ‘high-risk’ participants reclassified as ‘intermediate risk’ after the first BCSP surveillance colonoscopy was analysed using a log-binomial regression model. The risk difference and 95% CIs were presented.

• The number of participants with CRC detected prior to, or at, the first BCSP surveillance were to be summarised descriptively; however, there were no participants with CRC.

• The levels of RBC EPA and rectal mucosal EPA were summarised at baseline (RBC only) and at visits 4 (RBC only) and 6 for those receiving EPA-FFA and those receiving EPA-TG. In addition, the change from baseline to visits 4 and 6 were summarised.

• The levels of DHA, AA and the EPA-to-AA ratio were summarised at baseline (RBC only) and at visits 4 (RBC only) and 6 (RBC and rectal mucosal) for those receiving EPA-FFA and those receiving EPA-TG. In addition, the change from baseline to visits 4 and 6 were summarised for RBC samples.

• Dietary fish and other seafood intake (oily fish and total) at baseline (visit 1) and at the end of the trial (visit 6 or 7) were summarised by treatment group.

Safety analyses

Adverse events were summarised for the safety population, that is all participants who received at least one dose of trial medication. Summaries were based on the IMP that the participant received, irrespective of randomisation.

Both AEs and treatment-emergent ADRs were summarised by system organ class. GI AEs were also summarised by preferred term and by formulation. ADRs were summarised by severity using the preferred term. The worst case (i.e. severe and/or related to trial treatment) was assumed if severity or causality were missing, unless otherwise stated.

Clinically significant bleeding episodes (i.e. haemorrhagic stroke or acute GI bleeding requiring hospital admission or investigation) were identified by the chief investigator using a manual search of the full list of AEs and SAEs.

Common GI AEs associated with ω-3 PUFA and/or aspirin use were presented separately as clinically meaningful symptom categories, which were defined by the chief investigator.

Serious adverse reactions and ADRs that led to trial discontinuation were summarised by treatment group and preferred term.

All treatment-emergent ADRs were listed.

Primary outcome analysis

The test of interaction showed that there was no evidence of any interaction between EPA and aspirin for the ADRa (p = 0.85). Therefore, primary and secondary outcomes were analysed according to factorial margins, that is the treatment effects for EPA and aspirin were reported separately.

Table 17 shows adjusted risk differences of the ADRa for the treatment effect of EPA and aspirin. The point estimates and 95% CIs for both EPA and aspirin showed that there was no evidence of a statistically significant difference. These analyses were adjusted for whether or not the participant had a repeat endoscopic procedure (i.e. full colonoscopy, partial colonoscopy or FS) and included BCSP site as a random effect. Supportive analyses that were not adjusted by repeat colonoscopy showed similar results (see Appendix 4, Table 40).

The point estimates for EPA and aspirin were –0.9% and –0.6%, respectively, with 95% CIs that included zero, indicating no statistically significant difference from no treatment for both interventions. The trial was designed to detect an absolute ADRa difference of 10%. Figure 9 and Table 17 show that the lower limit of the 95% CIs did not reach –10% for either EPA or aspirin.

FIGURE 9.

Forest plot of the treatment effect of EPA and aspirin on the ADRa. The blue vertical line represents the prespecified target reduction of ADRa (10%). Adjusted by site as a random effect and by repeat colonoscopy at baseline.

Sensitivity analyses for the primary outcome

Several sensitivity analyses were conducted to investigate the robustness of the primary analysis. Although the point estimates and 95% CIs varied between different analyses, they were supportive of the primary analysis (Table 18 and Figure 10).

FIGURE 10.

Forest plots for the sensitivity analyses of the ADRa. (a) EPA vs. placebo; (b) aspirin vs. placebo; and (c) multilevel model. Results using the multilevel model are presented as odds ratios, whereas all other sensitivity analyses are presented using the risk difference. Adjusted by site as a random effect and by repeat colonoscopy at baseline. Some analyses were adjusted by further variables.

The multilevel model included both BCSP centre and site as random effects to account for sites embedded within the same BCSP (see Figure 10).

Number of participants with colorectal cancer detected prior to or at first surveillance colonoscopy

There was no report of any CRC detected at the 12-month surveillance colonoscopy or during the intervention phase of the trial.

Clinically significant acute upper gastrointestinal bleeding episodes

No haemorrhagic strokes were reported during the trial. A manual search of AEs and SAEs revealed six acute upper GI bleeding events that were considered by the chief investigator to be of clinical significance. These were ‘oesophageal haemorrhage’ (in the placebo EPA + aspirin group), ‘gastro-oesophageal reflux disease’ (in the placebo EPA + aspirin group) and ‘alcohol withdrawal syndrome’ (in placebo EPA + placebo aspirin group), which were all reported as SAEs, and ‘gastric haemorrhage’ (in the EPA + placebo aspirin group) and two cases of ‘melaena’ (in the EPA + placebo aspirin and placebo EPA + aspirin groups), which were recorded as AEs. It is possible that one of the cases of melaena (in a participant receiving EPA + placebo aspirin) was a SAE because the participant had been hospitalised, but this event was recorded specifically as an AE by the site. All tables reflect this categorisation.

Deaths

One death was reported during the trial. During contact made to arrange visit 5, site staff were made aware that a participant had died from bladder cancer, which was deemed to be unrelated to the intervention.

Protocol deviations

Protocol deviations were reported for between 64% and 73% of participants in each treatment group. Most deviations were judged to be minor, with the majority being trial visits outside the time window. The number and types of deviations appeared similar between treatment groups.

There were 11 deviations related to randomisation error (Table 37). Ten of these were as a result of participants being ineligible, and one participant was randomised prior to consent. None of these errors occurred in participants who were randomised to the placebo + placebo group. There was no reason to suspect that the deviation was related to the treatment group to which the participant had been randomised.

Colorectal adenoma size

Given the decrease in total colorectal adenoma number associated with EPA and aspirin treatment, colorectal adenoma size was analysed consistent with size analysis in previous RCTs of chemoprevention agents in FAP patients. 55,67 Colorectal adenoma size was summarised within each participant across each treatment group. Analyses were based on the within-participant mean value (Table 38).

The adjusted mean difference between the EPA and no EPA groups was –0.47 mm (i.e. the mean adenoma size was smaller in those receiving EPA than in those not receiving EPA). The mean adjusted difference between the aspirin and no aspirin groups was 0.42 mm (i.e. the mean adenoma size was bigger in those receiving aspirin than in those not receiving aspirin). There was no statistically significant difference between EPA and placebo users, or between aspirin and placebo users.

Relationship between individual colorectal adenoma number and eicosapentaenoicacid levels

The relationship between the change in RBC EPA levels at 12 months from baseline and secondary outcomes, according to EPA factorial margin, was also investigated descriptively by plotting individual values. There was no evidence of a clear relationship between individual increase in RBC EPA level and total colorectal adenoma number (Figure 19). This was also the case for the rectal EPA level at 12 months. The fact that there were three ‘outlier’ individuals in the placebo EPA group who had a large increase in RBC EPA level during the intervention phase suggests that ‘contamination’ by own-use ω-3 PUFA intake may have occurred in these cases (see Figure 19).

FIGURE 19.

Change in RBC EPA levels at 12 months from baseline against total number of colorectal adenomas per participant by EPA treatment groups. (a) Placebo EPA; and (b) active EPA.

Relationship between red blood cell and rectal mucosal eicosapentaenoic acid levels in participants

The relationship between RBC and rectal mucosal EPA levels at 12 months was of moderate strength, with a correlation coefficient of 0.455 (Figure 20).

FIGURE 20.

Scatterplot of the individual rectal mucosal EPA level against the corresponding RBC EPA level at 12 months.

Trial design

A key strength of the seAFOod trial was integration in the English BCSP, which continues colonoscopic surveillance in the BCSP, unlike in Scotland, where ongoing surveillance responsibility is passed to general endoscopy services. This provided several advantages to the trial, including:

• consistent high-quality colonoscopy performance and reporting above that available in general colonoscopy populations

• accurate screening colonoscopy data from the BCSP, with which to predict screening colonoscopy outcomes for recruitment projections

• uniform, protocol-driven care within the BCSP, particularly the small window for the 12-month surveillance colonoscopy in the BCSP, despite high procedural pressure in NHS endoscopy units

• use of a ‘high-risk’ surveillance cohort and 1-year colonoscopy outcomes, which minimised the intervention period compared with previous polyp-prevention trials,34–36,141,143 with consequent time and cost savings.

Another strength was the comprehensive seAFOod trial biobank with careful QA control. The biobank will be critical for further biomarker-driven, stratified analysis of the trial data. Availability of dietary and tissue biomarker data for the ω-3 PUFA intervention allowed interpretation of the EPA effect on colorectal outcomes, analysis of which could be confounded by dietary and OTC ω-3 PUFA exposure.

The small number of advanced colorectal adenomas found during the seAFOod trial is a limitation and probably relates to the short surveillance interval, as well as the excellent quality of the clearance screening colonoscopy. Previous polyp-prevention trials have reported an advanced ADRa of ≈10% in the placebo group of patient populations, approximating to combined ‘high-risk’ and ‘intermediate-risk’ BCSP patients with more heterogeneous follow-up (1–5 years). 34,36,141,143 Detailed data on advanced ADRa and MAP at surveillance colonoscopy were not available from the BCSP when the seAFOod trial was designed but are now available from the seAFOod trial and other reports98 in order to determine the feasibility of using advanced neoplasia as a primary or main secondary outcome.

As the first chemoprevention CTIMP in the BCSP, the outcome and performance data from the seAFOod trial will be invaluable for any future polyp-prevention trial (see Chapter 5, Recommendations for research). The seAFOod trial has established the BCSP as a realistic vehicle for interventional research during the endoscopy phase of the patient pathway.

Trial performance

Trial and data management: Nottingham Clinical Trials Unit

Lelia Duley, Professor of Clinical Trials Research; Alan A Montgomery, Professor of Medical Statistics and Clinical Trials and Deputy Director; Trish Hepburn, Senior Medical Statistician; Wei Tan, Medical Statistician; Dan Simpkins, Senior Data Manager; Anna Sandell, Trial Manager from November 2011 to 2014; Kirsty Sprange, Trial Manager from May 2014 to August 2015 then Senior Trial Manager from 2015; Sarah Fahy, Trial Manager from October 2015 to September 2016; Aisha Shafayat, Trial Manager from December 2016; Eleanor Harrison, Trial Administrator from October 2011 to January 2012; Margarita Carucci, Trial Administrator from February 2012 to July 2014 then Trial Co-ordinator from August 2014 to August 2015; Natalie Hutchings, Trial Administrator from September 2014 to June 2016; Robert Allen, Trial Co-ordinator from August 2016; Gill Bumphrey, Trial Pharmacist from March 2011 to June 2015; Sarah Walker, Data Co-ordinator from February 2013 to August 2017; Matthew Foster, Data Administrator; Chris Rumsey, Information Technology Programmer; Justin Fenty, Statistician from April 2011 to September 2012; and Veronica Moroz, Statistician from May 2009 to December 2010.

Co-applicants

Richard F Logan, Professor of Clinical Epidemiology, University of Nottingham; Colin J Rees, Professor of Gastroenterology and Consultant Gastroenterologist, Newcastle University and South Tyneside NHS Foundation Trust; Gayle Clifford, South of Tyne Bowel Cancer Screening Centre, BCSP, SSPr; Paul M Loadman, Professor of Pharmacokinetics and Drug Metabolism, University of Bradford and Yorkshire Experimental Cancer Medicine Centre; Anna Nicolaou, Professor of Biological Chemistry, University of Bradford; Devon Devonport, Patient and Public Representative (BCSP Patient Representative); Paul Silcocks, Senior Lecturer in Medical Statistics, University of Nottingham; and Hywel Williams, Professor of Dermato-epidemiology, University of Nottingham.

Trial Management Group

Professor Mark A Hull, Professor of Molecular Gastroenterology and Honorary Consultant Gastroenterologist; Diane Whitham, Associate Professor in Clinical Trials; Alan A Montgomery, Professor of Medical Statistics and Clinical Trials and Acting Director; Anna Sandell, Trial Manager; Eleanor Harrison, Trial Administrator; Wei Tan, Medical Statistician; Eleanor Mitchell, Senior Trial Manager; Kirsty Sprange, Trial Manager/Senior Trial Manager; Margherita Carucci, Trial Co-ordinator; Gill Bumphrey, Trial Pharmacist; Sarah Fahy, Trial Manager; Natalie Hutchings, Trial Administrator; Brian Barnes, Data Manager; Matthew Foster, Data Administrator; Aisha Shafayat, Trial Manager; and Robert Allen, Trial Co-ordinator.

Trial Steering Committee (independent members)

Professor Will Steward (Chairperson), Triallist and ex-Chairperson, National Cancer Research Institute CRC Clinical Studies Group, University of Leicester; Professor Greg Rubin, Triallist with primary care gastroenterology expertise, Newcastle University Institute of Health Sciences; Sally Benton, Director, NHS Bowel Cancer Screening Southern Programme Hub (previous Director: Professor Stephen Halloran); Mr Alan Reece, BCSP Patient ‘Service User’ Representative; Dr Elmar Detering, Representative of Bayer Pharma AG (Observer); and Mr Justin Slagel, Representative of SLA Pharma AG, until IMP switch (Observer).

Data Monitoring Committee (independent members)

Professor Bob Steele (Chairperson), Professor of Surgery, University of Dundee; Professor Dion Morton, Consultant, University Hospitals Birmingham NHS Foundation Trust; and Professor John Norrie, Chair of Medical Statistics and Trial Methodology, Director of Edinburgh Clinical Trials Unit, University of Edinburgh.

Institute of Cancer Therapeutics, University of Bradford

Professor Paul M Loadman, Professor of Pharmacokinetics and Drug Metabolism; Ms Amanda Race, Research Assistant; Ms Elizabeth Macken, Research Assistant; and Ms Jade Spencer, Research Assistant.

University of Sheffield

Dr Elizabeth A Williams, Senior Lecturer in Human Nutrition.

Trial sites

• North Tees and Hartlepool Hospitals NHS Foundation Trust: Matthew Rutter (PI), Debbie Wilson, Anne Eastick, Carol Adams, Susan Kelsey and Tracey Johnston on behalf of the site.

• Newcastle upon Tyne Hospitals NHS Foundation Trust: John Mansfield (PI), Heather Dixon, Elaine Stephenson, Maria Price, Mary Doona, Bev Douthwaite, Carrie Parker, Emma Crossland, Julie James, Lesley Dodd, Nicola George, Sasha Skentelbery and Sharron Lee on behalf of the site.

• Northumbria Healthcare NHS Foundation Trust: Tom Lee (PI), Mark Welfare (PI), Helen Bailey, Heather Dixon, Jane Dickson, Linda Patterson, Julie James, Lesley Dodd, Nicola George and Sasha Skentelbery on behalf of the site.

• Derby Teaching Hospitals NHS Foundation Trust: Andrew Goddard (PI), Maria Hartley, Helen Gibbs, Tracey Ambler, Beverley Powell, Christine Harrison and Keren Kerr on behalf of the site.

• Chesterfield Royal Hospital NHS Foundation Trust: Keith Dear (PI), Tracey Ambler, Carmel Cooke, Lucinda Wilson, Teri-Ann Sewell, Helen Gibbs and Maria Hartley on behalf of the site.

• Norfolk and Norwich University Hospital NHS Foundation Trust: Ian Beales (PI), Siobhan Parslow-Willams, Carmen Walker, David Tomlinson, Gemma Shearing, Jocelyn Keshet-Price, Jodie Graham, Kerrie Self, Melissa Rosbergen and Rachel Stebbings on behalf of the site.

• Calderdale and Huddersfield NHS Foundation Trust: Ashwin Verma (PI), Mandy Mellor, Bridget Keegan, Matthew Robinson, Natalie Austin, Philipa Gilbert and Stephanie Rich on behalf of the site.

• The Mid Yorkshire Hospitals NHS Trust: Syed Shah (PI), Lynsey Bourner, Annette Jones, Deborah Cooper, Jackie Ward, Patricia Kane, Rebecca Foster, Sarah Buckley, Steph Lupton, Thelma Darian, Toni Rank, Tracey Lowry and Bridget Keegan on behalf of the site.

• County Durham and Darlington NHS Foundation Trust: Clare Westwood (PI), Peter McCourt, Claire Shaw, Michelle Wood, Anna Archer, Emma Fenby, Gillian Matthews and Lynn Smith on behalf of the site.

• City Hospitals Sunderland NHS Foundation Trust: John Painter (PI), Pauline Oates, Verity Bennet, Hayley McMillan, Tracey Robson, Amanda King, Angela Hamilton, Dawn Charlton, Gayle Clifford, Mary Ritchie, Nicola Dempsey and Pamela Bowden on behalf of the site.

• Gateshead Health NHS Foundation Trust: Jitendra Singh (PI), Hayley McMillan, Verity Bennett, Ann Wilson, Sophie Gelder, Amanda King, Angela Hamilton, Dawn Charlton, Gayle Clifford, Mary Ritchie, Nicola Dempsey, Pamela Bowden, Sharon McCourt and Suzanne Nicholson on behalf of the site.

• South Tyneside NHS Foundation Trust: Colin J Rees (PI), Gayle Clifford, Carly Brown, Philippa Laverick, Hayley McMillan, Mary Ritchie, Nicola Dempsey and Verity Bennett on behalf of the site.

• Harrogate and District NHS Foundation Trust: Jonathan Harrison (PI), Barbara Heath, James Featherstone, Denise Cullingworth, Joanne Bell, Liz Potrykus and Pam Roth on behalf of the site.

• Leeds Teaching Hospitals NHS Trust: Richard Baker (PI), Ruth Fazakerley, Annette Jones, Doris Quartey, Felicia Onoviran, Claire Burton, Denise Cullingworth, Joanne Bell, Melissa Mellis, Nicola Bell and Pamela Roth on behalf of the site.

• York Teaching Hospital NHS Foundation Trust: James Turvill (PI), Nicola Broadley, Feveresterh Fallah, James Featherstone, Joanne Ingham, Julie Sackville Hamilton, Kay Kell, Paula Strider, Rebecca Coop, Denise Cullingworth and Elizabeth Potryrus on behalf of the site.

• Bradford Teaching Hospitals NHS Foundation Trust: Linda Juby (PI), Sophie Stephenson, Karl Ward, Philippa El Sayed, Rhian Simpson, Wendy Jepson, Hilary Bayton and Natalie Austin on behalf of the site.

• Airedale NHS Foundation Trust: Philip DaCosta (PI), Diana Wilkinson, Hilary Bayton, Jean Martin, Natalie Austin and Elleanor Waldron on behalf of the site.

• Northern Lincolnshire and Goole NHS Foundation Trust: Syed Muzaffar Ahmad (PI), Kirstie Smith, Ruth Loveday, Karen Martin, Kathy Dent and Sandra Evans on behalf of the site.

• North Cumbria University Hospitals NHS Trust: Frank Hinson (PI), Sara Underwood, Beverley Wilkinson, Christine Pearson, Jane Chester and Sue Meyrick on behalf of the site.

• Hull and East Yorkshire Hospitals NHS Trust: Graeme Duthie (PI), Bronwen Williams, Martin Lewis, Paula Brown and Sally Wood on behalf of the site.

• George Elliot Hospital NHS Trust: Edmond Sung (PI), Emiley Archer, Kerry Flahive and Linda Stretton on behalf of the site.

• Nottingham University Hospitals NHS Trust: Steve Foley (PI), Julian Williams (PI), Alison Large, Joyce Ntata, Karen Newcombe, Kathryn Moore, Sarah Chadderton, Chris Murfita and Shelley Biddles on behalf of the site.

• Northampton General Hospital NHS Trust: Udi Shmueli (PI), Ethelwolda Goyena, Andrea Jones, Caroline Duncombe, Elizabeth Tee, Jan Miles, Kathy McGrath, Mariska Pochin and Nancy Hopewell on behalf of the site.

• Kettering General Hospital NHS Trust: Andrew Dixon (PI), Joanne Walsh, Andrew Chilton, Margaret Turns, Maria Hill, Arrah Ashuarey and Ellen Nkhata on behalf of the site.

• University Hospitals of Leicester NHS Trust: John de Caestecker (PI), Alison Moore, Donna Ward, Elizabeth Andrzejewshi and Howard Fairey on behalf of the site.

• University Hospitals Coventry and Warwickshire NHS Trust: Jayne Eaden (PI), Steven Clay, Denise Gocher, Susan Dawson, Linda Stretton, Carol Wheatley, Kerry Flahive, Emily Archer, Katie James, Jaqueline Farmer and Jaqui Dagush on behalf of the site.

• South Warwickshire NHS Foundation Trust: Martin Osborne (PI) Kerry Flahive, Emily Archer and Lucy Hughes on behalf of the site.

• University Hospitals of Morecambe Bay NHS Foundation Trust: Colin Brown (PI), Jill Condor, Carmel Thomas, Carol Summer, Jane Chester, Maggie Coughlan and Susan Meyrick on behalf of the site.

• Poole Hospital NHS Foundation Trust: Sally Parry (PI), Diane Simpson, Tracey Deacy, Christine Dickson and Sarah Patch on behalf of the site.

• The Royal Bournemouth and Christchurch Hospitals NHS Foundation Trust: Sean Weaver (PI), Hannah Dewhurst, Emma Gunter, Ian Leadbitter, James Page, Nigel Butter, Nina Barratt, Maggie Bunce and Samntha Newman on behalf of the site.

• Dorset County Hospital NHS Foundation Trust: James Shutt (PI), Chris Hovell (PI), Abby Oglesby, Karen Hogben, Jackie Gibbins and Vicky Hanson on behalf of the site.

• Royal Surrey County Hospital NHS Trust: John Stebbing (PI), Karen Penry, Avril Adams, Daniel Jennings, Rachel Gallifent, Victoria Bartlett-Hunter, Anne Allen, Heather Swannack and Jessica Buckwell on behalf of the site.

• Frimley Park Hospital NHS Trust: Henry Tilney (PI), Karen Penry, Carrie Burgess, Jacqueline Brighton, Anne Allen, Heather Swannack, Jessica Buckwell and Rachel Gallifent on behalf of the site.

• Bolton NHS Foundation Trust: George Lipscomb (PI), Tracey Lawton, Janine Hurst, Jean Cummings, Karen Jewers, Louise Dawson, Shirley Cocks, Caroline Rogers and Gillian Cusack on behalf of the site.

• King’s College Hospital NHS Foundation Trust: Guy Chung-Faye (PI), Rayhan Ahmed, Stefi Stegner and Mun Lim on behalf of the site.

• Lewisham and Greenwich NHS Trust: John O’Donohue (PI), Abel Jalloh, Martha Handousa, Hazel Harrop, Katrina Armar, Laletha Agoramoorthy, Priscilla Phiri, Shanna Wilson, Stefania Stegner, Theodora Nago and Tim Soete on behalf of the site.

• Basildon and Thurrock University Hospitals NHS Foundation Trust: Javaid Subhani (PI), Anne Nicholson, Karen Steggles, Marco Bondoc, Anne Case and Sibongile Gorogodo on behalf of the site.

• University Hospitals Bristol NHS Foundation Trust: Tom Creed (PI), Shiney George, Edel Robbins, Jane Bowles, Jennifer Anstey and Nicola Barrah on behalf of the site.

• North Bristol NHS Trust: Melanie Lockett (PI), Ann Treasure, Carol Brain, Lisa Lillywhite and Suriya Kirkpatrick on behalf of the site.

• Torbay and South Devon NHS Foundation Trust: Rupert Pullan (PI), Sarah Tobin, Natalie Taylor, Kathryn Bird, Kerenza Boulton, Pauline Mercer and Georgina Ayres on behalf of the site.

• Plymouth Hospitals NHS Trust: Chris Hayward (PI), Sue Inniss, Ann-Marie Rowe, Judy Sercombe, Tim Johns and Kathryn Bird on behalf of the site.

• North West Anglia NHS Foundation Trust (Hinchingbrooke): Phillip Roberts (PI), Julie Maddocks, Lelsey-Anne Exon and Janet Jones on behalf of the site.

• North West Anglia NHS Foundation Trust (Peterborough): Naveen Kumar (PI), Julie Maddocks, Lelsey Ann Exon and Janet Jones on behalf of the site.

• Royal Cornwall Hospitals NHS Trust: James Bebb (PI), Keely Lane, Benita Adams, Fiona Hammonds, Heidi Duckworth, Helen Anderson, Lisa Trembath, Christine Taylor, Patricia Petrauske and Rebecca Warren on behalf of the site.

• Cheltenham General and Gloucestershire Hospitals NHS Foundation Trust: Simon Hellier (PI), Linda Hill, Estelle Nabela, Karen Holbrook, Natalie Bynorth, Paula Townshend, Sarah White, Sophie Delacruz and Christian Loveridge on behalf of the site.

• Royal Wolverhampton Hospitals NHS Trust: Matt Brookes (PI), Marie Green, Jayne Rankin and Jill Brown on behalf of the site.

• West Hertfordshire Hospitals NHS Trust: Alistair King (PI), Linda Sarginson, Elaine Walker, Olabisi Adeoti and Sarah Cerys on behalf of the site.

• Taunton and Somerset NHS Foundation Trust: Paul Thomas (PI), Jayne Foot, Leane Foote, Lucy Brotherton, Gillian Shire, Irene Cruickshank, Kelly Brown, Jennifer Williams, Julia Heneker and Karen Triggs on behalf of the site.

• Yeovil District Hospital NHS Foundation Trust: Steve Gore (PI), Katie Spurdle, Alison Lewis, Donna Haywood, Kate Ronaldson, Sarah Board and Suzette David on behalf of the site.

• Sherwood Forest Hospitals NHS Foundation Trust: Steve Foley (PI), Terri-Ann Sewell, Cheryl Heeley, Lynne Allsop and Dominic Nash on behalf of the site.

• Blackpool Teaching Hospitals NHS Foundation Trust: Mark Hendrickse (PI), Greta Duyvenvoorde, Rachael Wheeldon, Sue Hesketh, Louise Newton, Carly Hollin, Louise Johnson, Lucy Clarkson, Ruth Connelley, Sarah Strickland and Sylvia Taylor on behalf of the site.

• Lancashire Teaching Hospital Foundation Trust: Philip Shields (PI), Emma Durant, Ailsa Watt, Alexandra Williams, Janet Mills, Mark Verlander and Louise Newton on behalf of the site.

• Colchester Hospital University NHS Foundation Trust: Donagh O’Riordan (PI), Aine Turner, Alison Ghosh, Cathleen Chabo, Marianne Morgan, Natalie Wheatley, Nyasha Nago and Orla Thunder on behalf of the site.

• Ipswich Hospital NHS Trust: Simon Williams (PI), Joanne Bradley, Ginny Rose, John Cuckow, Stephanie Bell and Susan Cuckow on behalf of the site.

• East and North Hertfordshire NHS Trust: Peter McIntyre (PI), Elizabeth Green, Clare Collins, Nicola McNiff, Poppa de Sousa and Roisin Schimmel on behalf of the site.

• University Hospital Southampton NHS Foundation Trust: Phil Boger (PI), Emma Levell and Janet Jones on behalf of the site.