Polyposis registry clinicians recruited young male and female patients who met the following major eligibility criteria: An age of ≥ 10 and ≤ 21 years old and confirmed or a high likelihood of the presence of FAP. FAP status was determined by molecularly confirmed carriage of the FAP-associated APC mutation or by the determination of having a high probability of carrying the mutation on the basis of linked DNA markers or the presence of multiple colonic polyps, and/or multiple areas of congenital hypertrophy of retinal pigment epithelium in the context of a known FAP family history. Restricting eligibility to members of families manifesting classical FAP helped ensure homogeneity of disease in our study population. Current NSAID therapy, known aspirin sensitivity, major intercurrent illness, and pregnancy were grounds for exclusion. Advanced FAP was not a reason for exclusion unless surgery was planned within the next 12 months. The eligibility cut-off at 21 years old was based on concerns that continuation beyond this age might risk delaying preventive surgery. All patients were required to provide written consent to participate in the trial; written consent was required from the parents of patients under age 16 years. They were advised strongly to avoid all products containing aspirin or other NSAIDs and to use paracetamol for pain relief.
We conducted an international, multicenter, double-blind, randomized trial with four arms: aspirin (600 mg as 2 tablets/d) plus matched placebo, RS (30 g as 2 sachets/d in a 1:1 blend of potato starch and high amylose maize starch [Hylon VII]) plus matched placebo, aspirin plus RS, and placebo plus placebo; this trial employed a 2 × 2 factorial design. All polyposis registries and clinics in Europe were invited to participate in the CAPP1 Study. Our accrual goal was 208 patients, 52 in each of the four intervention arms.
Randomization was performed centrally at Leeds University via a blocked randomization scheme with a block size of 16 so that each set of 16 recruits contained 4 persons within each of the 2-×-2 trial arms. The blocked randomization was stratified at the level of geographical regions recruiting sufficient numbers of participants in order to avoid confounding of trial results due to geographical differences (e.g., regional diet); the regional strata were as follows: United Kingdom, Scandinavia, Northern Europe and southern Europe. Last, if siblings were enrolled, we assigned each member of such family groups the same combination to remove the potential for package mix-ups within the family. The duration of intervention was from 1 to a potential maximum of 12 years, with a scheduled annual clinical examination including endoscopy. Patients were advised of their option to leave intervention after each annual examination but were invited to remain on intervention (up to and including age 21 years).
The primary trial endpoint was the proportion of patients with an increased polyp count in the rectum and sigmoid colon after intervention. A major secondary endpoint was size of the largest polyp, which was chosen as another quantifiable and objective measure of disease severity. Given the multiple, international centers participating in this trial, it was not feasible to influence the clinical decision on whether the largest polyp was removed or left in situ nor to tattoo any polyps for review at a later examination. We also assessed crypt dimensions. Secondary laboratory endpoints included proliferative-state assessments of 1) crypt-cell proliferation (CCP) measured by direct counting of mitoses and of 2) indices of proliferation measured using antibodies against proliferating-cell nuclear antigen (PCNA) and Ki67.
The RS dose was based on the maximum easily tolerated doses and doses used previously in short-term intervention studies with CRC biomarkers as endpoints (19
). Aspirin and its matched placebo were supplied by Bayer (Leverkusen, Germany); the matched placebo was a lactose tablet (packaged at the pharmacy of Freeman Hospital in Newcastle). RS and its matched placebo were supplied by the National Starch and Chemical Company (Bridgewater, New Jersey); the matched placebo was 30 g/d of digestible corn (maize) starch. Children under 45 kg or under 12 years of age took half doses.
The trial was conducted in accordance with principles of the Declaration of Helsinki, and each study center was required to get the protocol, including the final version of the patient-information and informed-consent form, approved by its ethics committee.
Endoscopists counted the actual number of polyps in the rectum and sigmoid colon if there were 10 or fewer and provided an estimate (11–15, 16–20, 21–30, 31–50, 51–100, >100) when more numerous. We also collected data on the total number of polyps (rectum and sigmoid, ascending, transverse, and descending colon), which presented a challenge because of differing endoscopy policies and the challenge of the total number of polyps in an FAP patient, which can run into the thousands. When the study was initiated, many centers used sigmoidoscopy under sedation to monitor young people with FAP and usually avoided polyp removal unless a polyp became very large or appeared to be advanced. Some centers relied on paediatric endoscopists who tended to prefer full colonoscopy under anaesthetic. A diagrammatic form was used to clarify polyp-count (actual or estimated) instructions and to invite the endoscopist to mark the maximum extent of examination. The size of the largest polyp was recorded on the form (estimated by comparing the polyp with open 5-mm biopsy forceps).; We also requested all centers to record a withdrawal video of the rectum. If it was necessary to remove polyps, we asked that a recording be made before and after the removal. We specified rectal videos because we expected higher compliance among busy endoscopists with a rectal versus a more-comprehensive, more-time–consuming video. At least two withdrawal recordings in different perpendicular planes (i.e., at 90°) or with views taken from 10, 2, and 6 o’clock and starting at 20 cm from the anal verge were requested. At least twice during the recording, 5-mm forceps were opened near a polyp to assess the size of polyps and to provide a scale for the views. Almost all endoscopists complied with the request for open-forceps reference images, but there was considerable variation in extent of recording, with some offering a full-withdrawal recording rather than only imaging the rectum. The video recordings were scored (better, worse, or same) by two experienced endoscopists (SB and RKSP) blinded to intervention and the time point of examination (baseline, first year, second year, etcetera).
For assessing proliferative-state endpoints, six 5-mm mucosal biopsies were obtained from macroscopically normal mucosa from 5 to 15 mm from the anal verge; two biopsies were fixed in 75/25 absolute alcohol/glacial acetic acid for CCP, two were fixed in methacarn fixative (60 methanol:30 chloroform:10 glacial acetic acid) for PCNA immunostaining, and two in 10% neutral buffered formalin for determining Ki67 via MIB-1 immunostaining; these samples were transported to Newcastle University for processing. On receipt, CCP samples were transferred to 70% alcohol for long term storage.
Data and safety monitoring
Located in Newcastle University, the CAPP Operations Center housed the CAPP Principal Investigator (JB), Study Manager, Research Associate/Recruiter, technician, and pharmacist and coordinated the activities of the formal, independent CAPP Data Monitoring Committee (DMC) and the CAPP Steering Committee, which comprised multidisciplinary members to help in guiding the trial. The Operations staff coordinated day-to-day administration of the study, including dealing with study inquiries, distributing intervention packs, and collecting surveillance data including the hand-marked colorectal forms (which recorded the number and location of adenomatous polyps and size of the largest polyp in the examined colorectal segments), video recordings, and biopsy material for storage and processing. The Operations Center also offered study sites the option of genetic testing for the FAP-associated APC
mutation in samples obtained from prospective patients. All trial data were transmitted by the study sites to the Operations Center, where they were entered into a computerized study database and transmitted ultimately to the trial Statistical Center at Leeds University for analysis; prior to the end of study, only randomization data were held at the CAPP Statistical Center in Leeds. The CAPP Study Manager and principal investigator visited study sites frequently on an ad hoc basis, conducting chart reviews and other procedures necessary to ensure the integrity of the data and safety of patients. The CAPP statistical team at Leeds University annually reported the trial data to the DMC, chaired by Professor Doug Altman (London, Cancer Research UK), for safety and efficacy review. The DMC was responsible for interrupting the trial if and when warranted by statistically significant preliminary results. All of these operational procedures were facilitated by the long-standing collaborative association of our sites as members of a trials consortium called the Leeds Castle Polyposis Group, one of the two research communities which gave rise to the International Society for Gastrointestinal Tumours (InSiGHT – www.insight-group.org
To maintain interest in study participation, recruitment-site personnel were asked to contact each study patient at least once every six months to coincide with a delivery of study tablets and sachets to the patient. Unused tablets and sachets were collected, counted, and recorded by the recruiters to assess compliance with study interventions. The Operations Center sent study tablets and sachets once a month to the study sites so as to maintain regular, monthly contact with the sites; the Center also provided patients with regular updates of study progress to maintain enthusiasm.
We assayed proliferative states via three techniques (21
) so as to assure reliable results for this important aberration. We used a)
a crypt-microdissection method in tissue fixed in alcohol/acetic acid to provide estimates of CCP because this method is technically robust and b)
two methods that applied antibodies to sections of formalin-fixed tissue for identifying dividing cells marked by the antibodies against PCNA and Ki67. As described earlier in Methods, six variously fixed biopsies were taken from normal mucosa near the anal verge at the baseline and annual examinations. When required for histological analysis to determine CCP, samples were re-hydrated, stained with Schiff’s reagent, and microdissected to enable direct counts of total numbers (and location) of mitoses in whole crypts (22
). The numbers of mitotic cells in each tenth of the crypt by length were recorded; crypt compartments were numbered from 1 (base) to 10 (luminal surface). Formalin-fixed biopsies were paraffin-embedded and sectioned routinely prior to immunohistochemical staining by MIB1 to detect Ki67 (22
) and by PC10 to detect PCNA.
Crypt width and length were measured in the microdissected specimens and formalin-fixed tissue sections via a graticule in the microscope eyepiece and were standardized via a reference slide. Results from 10 crypts were averaged for each biopsy assessed.
The study was designed to detect a statistically significant difference in the proportion of patients with an increased polyp number in the rectum and sigmoid colon in the aspirin (versus the non-aspirin) group or in the RS (versus the non-RS) group at the end of intervention (versus at baseline). The total number of polyps in the rectum and sigmoid colon (prior to any polypectomy) at all endoscopies subsequent to baseline was the outcome measure, i.e., the dependent variable using a random effects model. Data for the total number of polyps seen in the rectum and sigmoid colon were available on all participants for all colonoscopies.
We included the secondary endpoint of total number of polyps throughout the colon (adjusted for the extent of endoscopy to account for the variable completeness of endoscopy) so as to make more complete use of collected data. Variability in local policy over extent of endoscopy meant that polyps were counted, or estimated when too dense, in differing numbers of colorectal segments. We allowed for this variability by adjusting models for extent at all endoscopies subsequent to baseline, including it as an independent variable in the random effects models. Prior to the random effects modelling, linear regression was performed so that total number of polyps at baseline endoscopy was the dependent variable and extent at baseline endoscopy was the independent variable. Adjusted values were calculated from the linear regression, i.e., we computed the difference between the observed number of polyps at baseline and the average number of polyps for that extent at baseline endoscopy. This adjusted number of polyps at baseline was then included in the random effects model as an independent variable.
This was a 2 × 2 factorial analysis that compared the two aspirin arms combined (aspirin plus RS and aspirin plus placebo) with the RS-plus-placebo and placebo-plus-placebo arms combined, and compared the two RS arms combined (RS plus aspirin and RS plus placebo) with the aspirin-plus-placebo and placebo-plus-placebo arms combined. We estimated an event rate of 40% in non-responding patients, i.e., that 40% of the patients in that group would have an increased polyp number (rectum, sigmoid colon) at the end of intervention, and an intervention effect of 50% fewer patients (i.e., a total of 20%) developing an increased number in the responsive intervention group, based on the average effect recorded in the published observational studies (23
). Due to the suggestion in some of these studies that the effect was greater with prolonged use, the present study was designed to allow participants to remain on study for as long as they could tolerate the interventions. The final sample size estimate of 208 patients, 52 in each of the four arms of the factorial design, was based on early data indicating that almost all patients had detectable pathology and was designed to detect the anticipated intervention effect with an 80% power and alpha level of 0.025.
Major, prespecified secondary analyses included the size of the largest polyp at the end of intervention and assessed in the subset of patients who remained on study for > 1 year; these patients were anticipated to be more likely to be fully compliant and to respond to an active intervention. Measurements of crypt length and width, hemicrypt cell count, and proliferation in normal mucosal biopsies also were secondary endpoints; these endpoints were considered exploratory since there were no prior data on potential effects on which to base power calculations.
Eight outcome measures were analyzed: three clinical endpoints (number of rectal and sigmoid polyps, largest polyp size [mm], and overall polyp number) and five exploratory translational endpoints (mean total CCP, crypt width, crypt length, the mean number of MIB1-positive cells per crypt, and the mean number of cells per hemicrypt). All but the last variable were log transformed before analysis; for log-transformed outcome measures, the exponent of the intervention coefficient was taken to indicate the ratio of the outcome measure in treated to untreated patients; a ratio of 1.0 indicates no differences between treated and untreated patients.
Analysis of variance (ANOVA) and Pearson Chi-squared tests were carried out to compare baseline measures. Associations between the baseline measures and age and sex were tested using two-sided t-tests, Chi-squared tests, and Spearman’s correlations. Spearman’s correlation coefficients were calculated for all pair-wise combinations of the continuous baseline measures. Differences in final largest polyp size between the four intervention groups were investigated using linear regression. Modeling involved estimation of random effects, linear models, and generalized estimating equation (GEE) logit models to identify main effects and possible interactions between intervention and time. The primary analyses compared the two arms containing aspirin with the two arms not containing aspirin and separately compared the two arms containing RS with the two arms not containing RS. The outcome measures in these models comprised all results obtained after entry endoscopy, and all models were adjusted for entry data and time on the intervention (years). In addition, the random-effects models for the total number of polyps were adjusted for age and extent, i.e., the proportion of the colorectum examined. The total polyp number at entry was adjusted for the extent of endoscopy via adjusted values from linear regression; in specific, we computed the difference between the observed number of polyps and the average number of polyps for that extent of endoscopy. This residual number formed the adjusted total of polyps at entry endoscopy. In analyses of numbers of polyps, the number of polyps which were removed at any endoscopy other than the last was added to the total number of polyps recorded at the following endoscopy.
We tested differences between interventions in results from video analysis with Fisher’s exact test. All analyses were carried out using STATA version 8 (StataCorp. 2003. Stata Statistical Software: Release 8.0. College Station, TX: Stata Corporation), and probabilities less than 0.05 were considered significant.