Title: An international, randomized trial of celecoxib versus celecoxib plus difluoromethylornithine in patients with familial adenomatous polyposis

Corresponding Author: Patrick M. Lynch, JD, MD, Department of Gastroenterology, Hepatology and Nutrition 1466, The University of Texas M.D. Anderson Cancer Center, P.O. Box 301402, Houston, Texas 77230-1402, USA. Phone: 713-563-4384, Fax: 713-745-1886E-mail: plynch@mdanderson.org .

Disclosures: None. No conflicts of interests exist.

Registration: The trial is registered at ClinicalTrials.gov, number N01-CN95040.

Grant Support: This trial It was funded by Visor, and . also Ssupported by the NIH/NCI under award number P30CA016672 and used the Biostatistics Resource Group. and used the Biostatistics Resource Group

Co-Authors:

Carol Burke, MD Gastroenterology and Hepatology, Cleveland Clinic Main Campus, Cleveland, OH 44195, USA

Robin Phillips, MBBS, MS, FRCS, The Polyposis Regsistry,St Mark’s Hospital, London, United Kingdom.St Mark’s Hospital, Gastroenterology and Emergency Surgery, London, United Kingdom

Jeffrey S. Morris, Ph.D., Department of Biostatistics, Division of Quantitative Sciences The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Rebecca Slack, MS, Department of Biostatistics, Division of Quantitative SciencesThe University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Xuemei Wang, M.S., Department of Biostatistics, Division of Quantitative Sciences, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Jun Liu, MS, Plastic Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Rebecca Slack, MS, Department of Biostatistics, Division of Quantitative SciencesThe University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Sherri Patterson, BS, OVP Cancer Prevention and Pop Science, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Frank Sinicrope, MD, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA

Miguel A. Rodriguez-Bigas, MD, FACS, FASCRS, Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Elizabeth Half, MD, Gatroenterology Department, Rambam Medical Center, Haifa, Israel

Steffan Bulow, MD, Hvidore Hospital, Denmark, UK

Andrew Latchford, MBBS, BSc (Hons), FRCP, MDThe Polyposis Regsistry,St Mark’s Hospital, London, United Kingdom., Gastroenterology Department at Derriford, Derriford, PlymouthUnited Kingdom

Sue Clark, MA, MB BChir MD FRCS, The Polyposis Regsistry,St Mark’s Hospital, London, United Kingdom.Department of Surgery and Cancer, St. Mark’s Hospital,Imperial College, London, United Kingdom

William A Ross, MD, MBA, Gastroenterology, Hepatology, and Nutrition, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Bonnie Malone, BA, Gastroenterology, Hepatology, and Nutrition, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Hennie Hasson, BSN, Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, Ohio, USA

Ellen Richmond, BSN, Gastrointestinal and Other Cancers Research Group, Division of Cancer Prevention, The National Cancer Institute, Bethesda, Maryland, USA

Ernest Hawk, MD, MPH, Division of OVP, Cancer Prevention and Population Sciences The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Keywords: POLYP, FAMILIAL ADENOMATOUS POLYPOSIS, CANCER, ADENOMA, CHEMOPREVENTION

Word Count: 5, 504

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Abstract:

Background & Aim: Although NSAIDs reduce colorectal adenoma burden in familial adenomatous polyposis (FAP), the utility of combining chemopreventive agents in FAP is not known. We conducted a randomized trial of celecoxib (CXB) versus CXB+diflouromethylornithine (DFMO) to determine the synergistic effect, if any.Methods: The primary endpoint was % change in adenoma count in a defined field. Secondary endpoints were adenoma burden (weighted by adenoma diameter) and video review of entire colon/rectal segments. Adverse events (AE) were monitored by NCI toxicity criteria. Results: 112 subjects were randomized:. sixty men and 52 women at with a mean age of 38 years.; For the 89 patients who had landmark-matched polyp counts available at baseline and 6 months, (ITT Measurable), the89 were evaluable for efficacy endpoints. The mean % change in adenoma count over the 6 months of trial was -13.0% for CXB+DFMO and -1.0% for CXB (p-value = 0.69). Mean % change in adenoma burden was -40% (CXB+DFMO) versus -27% (CXB) (p-value = 0.13). Video-based global polyp change was -0.80 for CXB+DFMO versus -0.33 for CXB (p-value = 0.03). Fatigue was the only significant AE, worse on the CXB arm (p=0.02). Conclusions: Celecoxib combined with DFMO yielded moderate synergy according to a video-based global assessment. No significant difference in adenoma count, the primary endpoint, was seen between the two study arms. No evidence of DFMO-related ototoxicity was seen. There were no adverse CV outcomes in either trial arm and no significant increase in adverse events in the CXB+DFMO arm of the trial. Differences in outcomes between primary and secondary endpoints may relate to sensitivity of the endpoint measures themselves.

Significance of this study

What is already known about this subject?

The cyclooxygenase-2 inhibitor, celecoxib, reduces adenoma burden in FAP patients by about 30%

The polyamine synthesis inhibitor, Difluoromethylornithine or DFMO, when combined with NSAID’s reduces tumors in animal carcinogenesis models

DFMO, when combined with sulindac, reduces risk of recurrent adenomas in patients with non-familial adenomas by about 70%

What are the new findings?

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Celecoxib combined with DFMO is well-tolerated in patients with FAP, with no irreversible hearing loss related to DFMO

As measured by our traditional measure, adenoma count in a defined area, the combination of celecoxib and DFMO yields only a nonsignificant improvement compared with celecoxib alone

A secondary, but arguably more valid measure of adenoma burden, based on global video assessment, does demonstrate a significant incremental benefit through the addition of DFMO to celecoxib

How might it impact on clinical practice in the foreseeable future?

Results of this randomized clinical chemoprevention trial in adults with FAP leave open the question of magnitude of benefit when adding DFMO to an NSAID. Because differences in outcome were importantly dependent on the method used to measure adenoma burden, even more careful attention to said methods will be critical in the interpretation of trial results in the future. Drug-approval processes that increasingly call for demonstration of “clinical benefit” underscore the importance of response measures.

Introduction

Familial Adenomatous Polyposis (FAP) is an inherited susceptibility to diffuse colorectal adenomas, and to colorectal carcinoma, occurring in close to 100% of unresected colons, and caused by a germline mutation in the APC gene located on the long arm of chromosome 5[1] . [2 3]. To prevent cancer development it is recommended that patients with FAP undergo colectomy with ileo-rectal or ileo-anal anastomosis at a time when polyp burden, size or degree of dysplasia are beyond the scope of safe endoscopic management[4]. This usually occurs during the 2nd or 3rd decade of life. Patients with the attenuated FAP phenotype, often associated with mutations at the 5’ terminus (exons 1- 4), have fewer polyps and can often further delay colectomy[5].

An effective chemopreventive agent or combination of agents with favorable toxicity profiles would be of substantial benefit to FAP patients. Benefitting most are those with an ileorectal anastomosis and recurrent rectal polyps, young adults with intact colon who wish to delay colectomy, as well as patients with duodenal adenomas. If effective in FAP, such a drug or drugs may also be efficacious in the much larger population of patients with sporadic adenomas who are at increased risk of colorectal cancer.

Aspirin and other conventional NSAIDs are associated with a decreased incidence of colorectal neoplasia but the risk of known side effects of this drug class diminish theirits utility as a chemopreventive agents. Selective COX-2 inhibitors were developed to minimize these toxicities, particularly gastrointestinal mucosal injury and its complications, a consequence of NSAID inhibition of COX-1.[6 7]. Celecoxib preferentially inhibits cyclooxygenase-2 (COX-2) over COX-1. COX-2 is an enzyme that is rapidly induced at the site of inflammation. Elevated levels of COX-2 are found in many pre-malignant lesions such as colorectal adenomas[8 9] . Experimental evidence suggests that COX-2 inhibitors inhibit angiogenesis and induce apoptosis [10]. These mechanisms may play an important role in the prevention or regression of colorectal polyps. Animal models also support the concept that COX inhibitors may play an important role in cancer prevention[6 11 12].

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COX-2 inhibitors have been shown to be effective chemopreventive agents in short term trials of both FAP[13 14] and nonfamilial adenomas[15-17]. However, significant cardiovascular adverse event signals arose from several of the longer term sporadic adenoma trials [18 19], leading to the removal of rofecoxib from the market and black-box warnings for celecoxib as well as nonselective NSAIDs.

Difluoromethylornithine (DFMO) is an irreversible, enzyme-activated inhibitor of ornithine decarboxylase (ODC), the key enzyme in mammalian polyamine synthesis, which acts through covalent binding to the enzyme[20 21]. By blocking the conversion of ornithine to putrescine, DFMO effectively inhibits the rate-limiting first step in the biosynthesis of polyamines [22]. Although the precise role of polyamines are not known, levels of ODC are closely related to tumor promotion, and inhibition of ODC is associated with suppression of tumor development in laboratory animals. DFMO was found to be an effective inhibitor of carcinogen-induced colon tumors in rats and also to inhibit cell proliferation and possibly mucosal prostaglandin levels[11 23-25]. Nonetheless the precise pathways by which ODC inhibition translates into tumor inhibition remains unclear. In comparison to various NSAIDs, DFMO significantly inhibits the progression of azoxymethane (AOM)-induced aberrant crypts in rat, though somewhat less effectively than sulindac or piroxicam[25]. DFMO has also been studied in animal models in combination with NSAIDs. Low dose DFMO and low dose piroxicam were found to have an additive effect in chemoprevention of AOM induced carcinomas in rats. The effect exceeded that of high dose piroxicam or high dose DFMO alone[11 26]. Efficacy of the combined regimen in inhibiting carcinogen-induced colon carcinogenesis in rats was substantiated in other studies in rodents[25 26]. Whether the effect of combined drug treatment reflects additive or synergistic mechanisms is not yet known. Under specific conditions ODC induction by bile acids or growth factors (EGF) may be prostaglandin-dependent. Such findings support the potential for synergistic chemopreventive effects by DFMO and NSAIDs such as Celecoxib.

DFMO has undergone Phase I and Phase II colon studies and studies focused on other organ sites

(cervix, bladder, prostate) and was found to be well tolerated at the dose of 0.5 g/m2/d for 10

months[27 28]). As a cancer chemotherapeutic agent, the major dose-limiting effects of DFMO have been thrombocytopenia, gastrointestinal toxicity, (nausea, epigastric pain, stomatitis, and diarrhea) neutropenia and reversible, dose-related ototoxicity[29] .

Experimental evidence indicates that a combination of chemopreventive agents is a very promising strategy against colorectal cancer. Studies in the Apc Min mouse indicate that combined treatment with the NSAID piroxicam and DFMO was more effective than either agent alone [30]. In another study, aspirin combined with DFMO had a synergistic effect against AOM-induced colon tumors in rats

The key study that bears on the trial reported here is that of Meyskens et al [31] in patients with nonfamilial adenomas. Using a combination of sulindac, 150 mg/d and DFMO, 500 mg/d, versus placebo, a dramatic (>70%) reduction in the rate of recurrent adenomas was observed a magnitude of effect greater than that achieved with any single agent to date.

Recognizing the polyp regressing action of celecoxib and the potential synergy with an agent

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acting by a different pathway (DFMO), we undertook a randomized clinical chemoprevention trial in adult FAP patients.

Methods

The trial was conducted at the University of Texas MD Anderson Cancer, the Cleveland Clinic in Cleveland and St. Mark's Hospital in Harrow, UK. Persons with FAP were recruited from the UT M.D. Anderson Cancer Center Hereditary Colorectal Cancer Registry, Cleveland Clinic David G. Jagelman Inherited Colon Cancer Registry and the St. Mark's Hoapital Polyposis Registry, and from outside referrals. Institutional Review Board (IRB) ethics panels approved the study protocol at each of the participating institutions. The trial was listed with the Clinical Trials Registry (www.clinicaltrials.gov ) . Eligible participants aged 18-65 years were required to have a clinical diagnosis of FAP based on a personal history of one of the following a) >100 adenomas; b) >10 adenomas before age 40 years; c) >25 adenomas and, if age >40 years, a characteristic family history (autosomal dominant pattern) including >100 polyps in a first degree family member, >25 polyps in two relatives in two generations, genetic diagnosis in a relative, or d) genetic diagnosis by sequencing or similar assay. Participants were required to have an evaluable colon and/or rectal segment, and 5 or more colorectal polyps’ ≥ 2mm at baseline examination. Additional eligibility requirements included: willingness to abstain from use of NSAIDs, including aspirin, for the duration of the study (cardio-protective dose of aspirin, > 80 mg, was permitted, following review/approval by PI), and agreement to use adequate contraception.

Exclusion criteria included: a) clinically significant hearing loss (defined as affecting everyday life or use of hearing aide) b) participants whose air conduction pure tone audiogram revealed a deficit differing from age specific norm by more than 30 dB averaged across two contiguous test frequencies in either ear; c) unwillingness or inability to sign informed consent; d) anticipated colectomy, within eight months of randomization, based on heavy polyp burden at baseline; e) history of hypersensitivity to COX-2 inhibitors, sulfonamides, NSAIDs or salicylates; f) chronic use of NSAIDs, including aspirin or celecoxib, at any dose during the six months prior to study entry (willingness to undergo a three-month washout period restored eligibility); g) use of fluconazole, lithium or chronic use of adrenocorticosteroids; h) history in the preceding year of discrete gastric or duodenal ulcer of size >5 mm (those with Helicobacter pylori related peptic ulcer became eligible for study upon successfully completing antibiotic treatment); i) invasive carcinoma in the past five years other than resected TNM T1-2, N0 colorectal cancer or resected non-melanoma skin cancer; j) partial or complete colectomy within 12 months prior to enrollment; k) inability to return for follow-up tests; l) significant medical or psychiatric problems, (including significant renal, hepatic or hematologic dysfunction) which would make the individual a poor protocol candidate; m) use of any investigational agent within the last 3 months, or at the discretion of the medical monitor; n) history of pelvic radiation; o) Hemoglobin <10.0 gm/dl, platelet count <100,000/ml; WBC with differential <3,000/ml; SGPT >1.5 x upper limit of normal, SGOT >1.5 upper limit of normal, alkaline phosphatase >1.5 x upper limit of normal, bilirubin >2 x upper limit of normal, creatinine >1.5 x upper limit of normal; p) positive serum pregnancy test within 14 days prior to baseline randomization; q) known or prior coagulopathy.

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Randomization details: This trial was designed as a double-blinded randomized trial to compare the percent reduction in polyp counts between patients treated with Celecoxib (CXB) with either DFMO (CXB+DFMO) or placebo (CXB+PBO) after 6 months of therapy. The design called for a total of 120 patients to be equally randomized, and divided in 1:1 ratio between to the two arms. A balanced randomization list was generated in RANLIST (https://biostatistics.mdanderson.org/SoftwareDownload/SingleSoftware.aspx?Software_Id=29) with random balance points every 4-14 patients, and this list was provided to the lead institution study pharmacist by the Department of Biostatistics from a statistician not associated with the analysis of the trial. The pharmacist kept this list in a locked cabinet. As patients were enrolled by the research nurse at the local site, the local site pharmacist would contact the lead site pharmacist for treatment assignment. The pharmacist at each institution was to maintain the confidentiality of the treatment assignment, dispense the assigned drug, and be the only entity that had access to unblinded status of individual patients during the course of the trial. The protocol provided for emergency access to this information by the PI, in the event of serious adverse event potentially related to drug. No such emergency access was called for during the trial period.

Endoscopic procedures: baseline on-study colonoscopy or sigmoidoscopy as well as off-study examinations were performed with standard videoendoscopes. Video of entire withdrawal was recorded. Still photos of informative reference polyps clusters were captured. When such clusters were not adjacent to an existing anatomic landmark, such as appendiceal orifice, ileocecal valve, surgical anastomosis, or retroflex view of rectum, an India ink tattoo was placed and photographed with the polyp cluster. Polyp diameters were estimated with open or closed biopsy forceps.

Polyps were measured and reported in three ways. First, and corresponding to the primary endpoint, total polyps were counted in defined anatomic fields, utilizing still-color photographs, before and after treatment. As a secondary endpoint, we calculated polyp burden. Polyp diameter was recorded in size categories: 0-1, 1-2, 2-3, 3-4, 4-6 and >6mm. The total polyp burden for each patient was calculated by multiplying the number of polyps in each size category by the corresponding median polyp size for that category (or actual diameter for polyps > 6mm) and then summing over all the size categories in all matched regions.

Another secondary endpoint was a video rating of global polyp burden at 6 months compared to baseline. Five independent reviewers compared paired videos while blinded to order (pre- vs. post treatment) as well as to treatment arm. The first video in the pair could take the value of -2 (much better), -1 (better), 0 (the same), 1 (worse) or 2 (much worse) relative to the second video. Videos from each of four regions (cecum-ascending, transverse, descending-sigmoid, rectum) for each patient were separately scored and then averaged for patients with intact colons, and only the rectal segment was scored for post-colectomy subjects.

Interventions were as follows: 1) Celecoxib, 400 mg p.o. BID plus DFMO placebo; or 2) Celecoxib, 400 mg p.o. BID plus DFMO 0.5 gm/m2/day rounded down to the nearest 250mg dose (BSA of < 1.4 = 500mg/day; BSA of 1.5 - 2.0 = 750mg/day; BSA of 2.1 - 2.5 = 1000mg/day; BSA of > 2.6 = 1,250 mg/day).

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In addition to the primary efficacy endpoint, the primary safety endpoint was relative tolerability and safety of Celecoxib + DFMO.

Patients who dropped out after study drug administration due to adverse events or other reasons were not replaced. Those exiting the trial early were encouraged to complete the end-of-study procedures.

Following reports of COX-2 inhibitor-associated cardiovascular toxicities, the trial was suspended from 17 December 2004 to 18 March 2005, pending reevaluation of cardiovascular risks. At one site the trial was terminated at this point, as IRB approaval was not gained for it to be resumed. However Tthe trial resumed at the other two sites, following the addition of exclusion criteria intended to address these issues: a) elevated C-Reactive Protein or CRP (> 3.0 mg/L); b) history of cardiovascular diseases or risk factors that might include one of the following: myocardial infarction, angina, coronary angioplasty, congestive heart failure, stroke, or coronary bypass surgery; c) uncontrolled hypertension (>135/>85 mm Hg on three repeated measurements during the 6 weeks prior to enrollment on the study). Eligibility was considered restored following successful treatment of known hypertension. Subjects with hypertension established for the first time at study entry were otherwise evaluated for protocol, randomized if they agreed to be monitored for BP, and were allowed to remain on-study for three months while undergoing antihypertensive therapy and monitoring. If, at the end of 3 months, subjects could not demonstrate successful BP control as measured and documented locally, dosing was suspended. Such subjects were nevertheless urged to complete 6-month offstudy evaluation, for intention to treat analysis; d) uncontrolled diabetes (subjects with established or new diagnosis were managed in a fashion similar to that for hypertensives, above; e) uncontrolled hypercholesterolemia (subjects with established or new diagnosis managed in fashion similar to that for hypertensives and diabetics, above—following the updated National Cholesteraol Education Program Adult Treatment Panel III Guidelines); f) family history of premature coronary disease (i.e. onset <55 years of age); g) metabolic syndrome diagnosis; h) history of deep venous thrombosis, pulmonary embolism, systemic lupus erythematosis, family history of protein S or C deficiencies, prior heparin-induced thrombocytopenia, Factor V Leiden deficiencies or high homocysteine levels; i) any indications for aspirin

Statistical methods. This trial was designed to compare the percent reduction in polyp counts between patients treated with Celecoxib (CXB) with either DFMO (CXB+DFMO) or placebo (CXB+PBO) after 6 months of therapy. A total of 120 patients were planned for enrollment in order to detect polyp count reductions of 41% from baseline to 6 months for CXB+ DFMO patients compared to 28% for CXB+PBO patients. Assuming 108 of these would be evaluable (54 per group), this would provide 80% power to detect this difference assuming a standard deviation of 24% and a 2-sided 5% significance level. An interim analysis was planned when 60 (50%) of patients completed the trial. Using O'Brien-Fleming stopping boundaries[32], the two arms would be declared to be different if the p-value was less than 0.003 or 0.049 in the interim and final analyses, respectively, preserving the overall 5% significance level.

A balanced randomization list was provided to the lead institution study pharmacist by the Department of Biostatistics from a statistician not associated with the analysis of the trial. The pharmacist at each institution was to maintain the confidentiality of the list, dispense drug based on this assignment, and be the only entity that had access to unblinded status of individual

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patients during the course of the trial.  The protocol provided for emergency access to this information by the PI, in the event of serious adverse event potentially related to drug.  No such emergency access was called for during the trial period.

Polyp counts and polyp burden scores from baseline and 6 months (or end of study procedures) were matched at up to 4 locations in the colon (cecum-ascending, transverse, descending-sigmoid, and rectum) for individuals with an intact colon or in the rectum for those with an IRA. Only fields with corresponding photos at each time point were used to calculate the percent change. As a result, for some patients their existing polyp counts at baseline were in fields not observed at 6 months, and thus they would have had a zero polyp count at baseline for the primary analysis. To accommodate this in the percent reduction calculations, the polyp counts were all increased by 1, at both baseline and 6 months, before calculating the percent change for analysis. For graphing, however, the percent change was not adjusted by adding 1 in order to maintain a meaningful visual scale.

The modified intent-to-treat (ITT Measurable) population includes all patients who had polyp counts and polyp burden scores available at both baseline and 6 months (or end of study). If the 6-month or end–of-study polyp count was missingnot available (see paragraph above),; subjects were assigned a change of 0% for the primary ITT (ITT All) analysis population. The evaluable population includes all patients with complete polyp counts and who completed 80% of treatment both overall and during the final 60 days of scheduled therapy, as defined in the protocol. This was defined in the protocol since CXB and DFMO are short acting ageents and it would be possible for patients to meet the 80% dose requirement but have no treatment for the weeks before the month 6 measurement. Patients for all 3 study populations were analyzed according to randomized treatment arm regardless of the actual treatment received.

For video scores, we calculated the mean rating score from the five reviewers., accounting for order. The mean rating scores for each region were calculated and the overall video rating score averaged for a single score per person.

Wilcoxon’s rank sum test was used to compare the counts of polyps at least 2mm by photos since diagnostic plots indicated significant right-skewness in the data, while t-tests were used to compare the polyp burden by photos and polyp burden by video between the two arms. Waterfall plots were created for each arm for the percent reduction in polyp counts for the ITT Full population, including all polyps.

For adverse events, specific symptoms were tabulated for all toxicities due to therapy that were experienced at grade 3 or higher or were experienced by at least 5% of patients, including only the highest grade per symptom per patient. For supplemental reporting, each patient’s highest grade was identified and tabulated by grade and treatment arm, regardless of symptom. Additionally, the incidence of highest toxicity grades was reported by treatment arm (as many times as the symptom occurred). These were reported overall and separately for symptoms related to study drug.

One of the known toxicities of DFMO is hearing loss, so this was carefully monitored in this trial. Eight hearing frequencies (250 Hz, 500 Hz, 1000 Hz, 2000 Hz, 3000 Hz, 4000 Hz, 6000

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Hz, and 8000 Hz) were tested in each ear. The variable of interest is the change of hearing threshold in decibels (dB) between baseline and 6 months. Characterizing this variable as continuous, we calculated the mean (SD), median, minimum and maximum values for each arm, and fit linear mixed models to perform a joint analyseis of all frequencies.

Results

Enrollment. 112 patients were randomized between December 2001 and August 2008. The study was closed at this point due to slow recruitment and with enrollment target almost met, with the mutual agreement of the DSMB, sponsor (NIH), and recruiting centres. At one point in timethat time, one site was found to have inadvertently left the treatment assignments unsecured, so technically therefore this site can no longer being considered double blind. All of those who performed photo and video scoring, however, were completely blinded the entire time.. The trial was closed and cannot be considered double-blinded at that site. For sensitivity, the data were summarized separately by institution, and are presented in a supplementary table. Analyses with and without patients from the unblinded institution were performed. , with consistent results. Results from all patients are presented here while results separated by instution are included in supplemental tables.

Counts of patients from randomization to evaluable status are presented in Figure 1. Table 1A presents the patient characteristic information by treatment arm. There was good balance between the treatment arms for gender, race, and institution. The placebo arm (CXB+PBO) had more patients who were diagnosed by the presence of more than 10 polyps before age 40 and more polyps at baseline overall. The CXB+DFMO arm had more patients who were diagnosed on the basis of > 25 polyps after age 40 and more patients with intact colon. Several differences exist among the patients from different centers (Supplemental Table S1). For example, St. Mark’s site 32 only enrolled patients with colectomies and tended to enroll older patients. Site 23Cleveland Clinic only enrolled white patients. Site 1 MDACC is the only institution that enrolled patients who met the FAP definition due to >25 polyps over age 40 with positive family history, and also enrolled patients with more polyps at baseline.

[32]Table 1.Patient Characteristics by Treatment Arm

All CXB+DFMO CXB+PBOCharacteristic N (%) N (%) N (%) P-valueAll Patients 112 (100) 57 (100) 55 (100)Age - median (min-max) 38 (18-63) 38 (19-59) 38 (18-63) 0.89

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Sex 0.84 Male 60 (54) 30 (53) 30 (55) Female 52 (46) 27 (47) 25 (45)Race 0.62 White 95 (85) 49 (86) 46 (84) Hispanic 11( 10) 4 (7) 7 (13) Black 1 (1) 1(2) 0 (0) Other 5 (4) 3 (5) 2 (4)Institution 0.91 MDACC Site 1 63 (56) 31 (54) 32 (58) Site 2 27 (24) 14 (25) 13 (24) Cleveland Site 3 22 (20) 12 (21) 10 (18) St. Marks 27 (24) 14 (25) 13 (24)Basis of FAP Diagnosis 0.36 >100 Polyps 44 (39) 23 (40) 21 (38) >10 polyps and age <40 51 (46) 23 (40) 28 (51) >25 polyps and age > 40 17 (15) 11 (19) 6 (11)Colon vs Rectum 0.17 Colon 46 (41) 27 (47) 19 (35) Rectum Only 66 (59) 30 (53) 36 (65)Number of Patients with Baseline Screen 108 (100) 57 (100) 51 (100)Number of landmark polyps at baseline screen 0.20 0-1 8 (7) 5 (9) 3 (5) 2-4 19 (17) 12 (21) 7 (13) 5-9 40 (36) 21 (37) 19 (35) 10 or more 41 (37) 19 (33) 22 (40)Number of landmark polyps at least 2 mm at baseline screen

0.05

0-1 33 (29) 20 (35) 13 (24) 2-4 35 (31) 23 (40) 12 (22) 5-9 30 (27) 8 (14) 22 (40) 10 or more 10 (9) 6 (11) 4 (7)Number of landmark polyps at least 2 mm at baseline screen for evaluable polyps in evaluable patients*

0.39

0-1 22 (32) 13 (37) 9 (27) 2-4 24 (35) 13 (37) 11 (33) 5-9 18 (26) 5 (14) 13 (39) 10 or more 4 (6) 4 (11) 0 (0)

*To be evaluable, patients had to 1) have matching polyp data at baseline and six months as well as 2) have completed at least 80% of the treatment both overall and for the final 60 days. Note: Every patient met the eligibility requirements for polyp count, however, those polyps were not required to be in matched landmark areas.

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Polyp Change. A total of 89 patients had complete polyp information. Table 2 summarizes the polyp regression results by treatment group using three outcomes: (1) percent change in polyps of at least 2mm, (2) percent change in total polyp burden, considering both size and number of polyps, and (3) change in polyp burden based on videos. The first two measures are quantitative measures based on the still photos, while the third measure is qualitative, but based on entire colonoscopy video that more comprehensively surveys the colon. These outcomes and the various analysis groups (ITT measurable, ITT all, and evaluable) are defined in the methods section.

Table 2. Response by Treatment Arm CXB+DFMO CXB+PBO

Population N Mean (SE) N Mean (SE) P-valuePolyp Counts from Photos including Polyps ≥2mm* ITT All 57 -0.11 (0.08) 55 -0.01 (0.11) 0.76 ITT Measurable 45 -0.13 (0.10) 44 -0.01 (0.14) 0.69 Evaluable 35 -0.08 (0.12) 33 0.10 (0.18) 0.46Polyp Burden from Photos ITT All 57 -0.32 (0.05) 55 -0.22 (0.05) 0.17 ITT Measurable 45 -0.40 (0.06) 44 -0.27 (0.06) 0.13 Evaluable 35 -0.41 (0.07) 33 -0.23 (0.08) 0.08Improvement from Video Ratings ITT All 57 -0.49 (0.1) 55 -0.26 (0.11) 0.13 ITT Measurable 35 -0.80 (0.14) 42 -0.33 (0.15) 0.03 Evaluable 28 -0.93 (0.15) 31 -0.36 (0.17) 0.01

ITT All = all patients – if the 6-month polyp counts are missing, then the patient is given a percent change of 0 per protocol description.ITT Measurable = All patients with baseline and 6 month polyp counts.Evaluable = ITT Measurable patients for that outcome who also took 80% of the treatment, both overall as well as during final 60 days.Evaluable = Patients with baseline and 6 month information who also took 80% of treatment overall and during final 60 days for photo data (counts and burden) or video rating.Video Ratings were qualitatively assessed on a 5 point scale from Much Better (-2) to Much Worse (2) by 5 reviewers whose scores were averaged across each assessed section of the colon. * Wilcoxon’s rank sum test was implemented instead of the t-test for the reduction in counts of polyps ≥2mm since data were skewed right. Results are similar for the t-test and Wilcoxon’s rank sum for these counts. The CXB+DFMO group had a mean reduction of 13% (SE=10%) in the number of polyps at least 2mm, while the CXB+PBO group had a mean reduction of 1% (SE=14%) (p=0.69). This difference was similar for evaluable patients or when missing polyp information was replaced with 0% differences (the full ITT All analysis). Total polyp burden was decreased by an average of 40% (SE 6%) from baseline to 6 months in the CXB+DFMO groups, and decreased by 27% (SE 6%) in the CXB+PBO group (p=0.13). This difference remained similar in the ITT All and evaluable analyses. In the video-based analysis, patients demonstrated greater improvement (-0.80 [SE 0.14]) in the CXB-DFMO arm compared to CXB+PBO (-0.33 [SE 0.15]; p=0.03). This difference was attenuated in the ITT full All analysis, but retained in the evaluable analysis (p=0.01). Figure 2 presents the percent reduction in all polyp counts for each patient ordered from largest increase to greatest reduction. The red line shows a reduction of 28%, the hypothesized average reduction for the CXB+PBO arm. We saw slightly more patients with elimination of all polyps in the CXB+DFMO group, and more patients with increasing

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polyp counts in the CXB+PBO group. Supplemental tables present these results separately by institution (Table S2A).) and with the potentially unblinded institution removed (Table S2B). The potential unblinding at Site 2 was investigated, and there was no evidence that the unlocked information left the pharmacy of that institution. The supplemental tables show that all measures have the same trends in all centers, but with a larger magnitude of difference between arms in Site 2, primarily with patients on CXB+PBO doing worse. In spite of this, the videos were assessed in a fully blinded fashion at a centralized location, so were not likely affected by the potential unblinding. One might argue that this larger difference may be due to an unblinded investigator tampering with measures. However, the videos were assessed in a fully blinded fashion at a centralized location so they are a good measure of whether these larger differences are a function of the site, or the potential unblinding

Toxicity. Adverse event information was included for all patients except one with missing information. Of the remaining 111 patients, 10 were documented to experience no toxicities on the trial. The counts of toxicities related to therapy are presented in Table 3 for any symptoms that occurred in at least 5 percent of patients or for which at least one patient experienced a grade 3 toxicity. The most common symptom was mucositis/stomatitis affecting 15 and 11 patients in the CXB+DFMO vs. CSBCXB+PBO, respectively. All grade 3 events related to study drug occurred on the CXB+DFMO arm and included one each diarrhea, gout, and high frequency hearing loss. The only symptom that differed significantly between the two arms was fatigue, which was worse in the CXB+PBO arm (p=0.02). High frequency hearing loss was of concern for patients receiving DFMO. It occurred in 7 patients on the CXB+DFMO arm compared to 4 patients on the CXB+PBO arm (p=0.53). Other common symptoms included diarrhea, heartburn/dyspepsia, nausea/vomiting, and headache.

Table 3. Numbers of Patients Experiencing Adverse Events Attributable to Study Drug*CXB+DFMO

N=56**Grade

CXB+PBO N=55Grade P-value

1 2 3 Total 1 2 3 TotalAuditory/Ear High Frequency Hearing loss 5 1 1 7 4 0 0 4 0.53

Constitutional Symptoms

Fatigue 2 1 0 3 8 3 0 11 0.02

Gastrointestinal Diarrhea 2 1 1 4 6 0 0 6 0.53

Heartburn/Dyspepsia 1 1 0 2 4 0 0 4 0.44

Mucositis/Stomatitis 14 1 0 15 8 3 0 11 0.50

Nausea/Vomiting 5 2 0 7 6 0 0 6 >0.99

Musculoskeletal Gout 0 0 1 1 0 0 0 0 >0.99

Pain Headache 1 0 0 1 5 0 0 5 0.11*This table includes only adverse events that occurred in at least 5% of the patients or a patient exhibited at least 1 grade 3 toxicity. Supplemental Table S3A includes all related toxicities by symptom category.

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** Adverse event information was included for all patients except 1 with missing information. Of the remaining 111 patients, 10 were documented to experience no toxicities on the trial. The p-value is comparing the overall proportion of patients experiencing each toxicity for each treatment arm, ignoring grade.

Additional toxicity tables are presented in Supplemental Tables S2A-S2E examining grades of symptoms and relation to study drug, disease, or other, regardless of specific symptoms. Of the 10 patients with documentation of no symptoms, 8 were in the CXB+DFMO arm compared to two in the CXB+PBO arm (p=0.09) whereas 8 patients had grade 3 toxicities in CXB+DFMO compared to 4 patients in the CXB+PBO arm (p=0.36). A total of 62 patients had any symptoms related to study drug, 30 vs. 32 for CXB+DFMO vs. CXB+PBO, respectively.

Among the total 206 drug-related toxicities, 92 (45%) of them occurred in the CXB+DFMO arm and 114 (55%) occurred in the CXB+PBO arm. Nineteen (48.7%) of the 38 drug-related grade-2 toxicities and all 3 (100%) drug-related grade-3 toxicities belong to the CXB+DFMO arm. The heterogeneity in reported toxicities, their infrequency, and lack of any consistent pattern of attribution makes it unlikely that any meaningful difference in adverse events exists between the two arms of the trial so only related toxicities are discussed further.

Symptom categories are presented in more detail in Supplemental Tables S3A and S3B. The most common categories with total counts for CXB+DFMO vs. CXB+PBO, respectively, are gastrointestinal symptoms (15 vs. 22), auditory/ear (10 vs. 14), constitutional symptoms (5 vs. 12; p=0.04), pain (3 vs. 5), and musculoskeletal/soft tissue symptoms (3 vs. 1). The distributions of symptom categories are similar across treatment arms, except for constitutional symptoms, with more events among patients receiving placebo. Any trends are in the direction of more symptoms in the CXB+PBO arm, but none are significantly different. The specific symptoms for these top five categories are specified in Table S3B.

In light of concerns for possible ototoxicity, hearing thresholds and related measures were evaluated as toxicity measures in their own right. We performed the Wilcoxon rank-sum test and found no significant difference between the two arms at any frequency level in either ear. Overall, for the left ear, there were 2 patients (8 observations) in the CXB+DFMO arm (patient # 28: at 1000 Hz, 2000 Hz, 3000 Hz, 4000 Hz, 6000 Hz and 8000 Hz; patient #21: at 4000 Hz and 6000 Hz) who had an increase of more than 25 dB in the hearing threshold between baseline and 6 months, compared to none in the CXB+PBO arm; and for the right ear, there was 1 patient in the CXB+DFMO arm (patient # 28: at 8000 Hz) and one patient in the CXB+PBO arm (patient # 62: at 250 Hz) who had an increase of more than 25 dB in the hearing threshold between baseline and 6 months.

In order to simultaneously assess the effects of treatment and frequency, a linear mixed-effect model was fit on the dB change between baseline and 6 months, using frequency, treatment and the interaction between frequency and treatment as covariates. Separate analyses were conducted for the left and the right ear. The analytic results showed no significant interaction between treatment and frequency for both ears, and no significant main effect due to treatment (p = 0.91, left ear and p = 0.27, right ear) or frequency (p = 0.39, left ear and p = 0.61, right ear).

Discussion

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We report findings that build upon an earlier randomized, placebo-controlled NCI sponsored study in FAP study participants evaluating the chemopreventive efficacy and safety of Celecoxib (100 or 400mg BID, respectively) versus placebo administered continuously for six months. In that study, 83 participants from two institutions were randomized to one of three arms and 78 (94%) participants completed the study. The primary efficacy variable was the percent change from baseline in the number of colorectal polyps. Twenty-three of thirty participants in the high dose arm had a reduction in their colorectal polyp burden, an average reduction of 28% overall. The findings were sufficiently encouraging as to support the study of this agent in combination with DFMO (Difluoromethylornithine).

The results of this study are equivocal insofar as the potential benefits of combining DFMO with celecoxib in the treatment of FAP. The primary endpoint, adenoma count in a defined region of the colorectum, did not yield evidence of any significant synergies. However, when the secondary endpoints of adenoma burden as measured by count x diameter and global change by video assessment were considered, significant or marginally significant improvement was observed. In our original trial of celecoxib versus placebo, significance was seen with each of the endpoint measures.

The differences in the video review scores per site do not showed consistent results among centers. Although at one site the treatment assignments were inadvertently left unsecured by the pharmacist, technically rendering this site no longer double- blind one, it should be emphasized that the trialists were not aware at any time of the randomization, and this was unlikely to have any impact on the scoring of photos or videos. Furthermore The photo reviews were done by an investigator at another site and the video reviews were performed by a panel, including experts not from the recruiting centres. The order in which the videos were played (ie baseline or end of study) was also randomized as to which was played first. Therefore we feel that the integrity of the video review and the trial were maintained.

What might account for the differences between the two studies? For one, the overall adenoma burden at baseline was less striking in the subjects participating in this trial, compared with the original trial. This may account for the lesser treatment effect seen in the CXB+PBO arm of this trial. However, it seems to be the case that DFMO does not afford a clinically dramatic effect when combined with celecoxib according to any of the measures employed to detect a treatment response.

Notwithstanding the modest nature of any possible incremental benefit from the addition of DFMO to celecoxib, it may nevertheless be appropriate to take a hard look at the way in which treatment response is measured in FAP. While we were disappointed that our primary endpoint, polyp burden in a small, defined region, failed to reach statistical significance, we have come to regard this as a suboptimal endpoint for several reasons. Treatment response by an ostensibly quantitative measure of adenoma count in a defined area suffers from several deficiencies. It is critically dependent on comparable technique being employed in capturing an identical field of polyps. Modest variation in technique from exam to exam is difficult to eliminate in GI endoscopy. Although such effects are randomly distributed between the two arms in a blinded trial such as this, they nevertheless introduce noise and thus tend to reduce the power to detect a

15

difference. This effect is magnified by a lower baseline polyp burden as experienced in this trial. We believe that these factors contribute to the more positive effect seen when more of the total available information is employed, as when measuring not only polyp count but also their diameter.

When a more global assessment is performed, as with our video scoring, all of the polyp data are available for consideration, even if not quantified as rigorously. Use of this method, we believe, is more clinically relevant in any event, and probably should have served as the primary endpoint in this trial. As digital video capture, video management (as with modern video editing programs), and on-line streaming for convenient scoring become more routinely employed, we anticipate use of entire colonoscopy findings to be the measure of response in future trials. Further, wWe are finding that use of a web-based program for quantifying adenoma burden can be used by multiple observers to conveniently and quickly score adenoma burden (sum of count ×X diameter) from colonoscopy videos in the clinical trial setting[33]. As importantly, the FDA has made it clear that endpoints in the treatment of FAP must meet a measure of “clinical benefit”, not merely some arbitrary percent reduction in adenoma burden. In order to satisfy such regulators as the FDA, we are developing a staging system for colorectal adenoma burden in FAP in which incrementally more aggressive interventions are tied to a given stage. In this fashion, measures such as delay in time to progression will, it is hoped, serve as a more clinically relevant endpoint for treatment efficacy. At the time of this writing, a validation study of such a staging system is nearing completion. It is anticipated that this will be a helpful part of the framework for future drug trials and will otherwise provide a common language for clinicians to employ in marking progression of severity of FAP polyp burden.

Overall, differences in adverse event rates between the celecoxib only and celecoxib plus DFMO arms in this trial did not appear to be clinically or statistically significant. Because our patient population was relatively young and the trial was short in duration, we not find or expect to find any evidence of cardiovascular toxicity in either arm of the study. Midway through the trial a careful reassessment of eligibility and monitoring was necessitated following the reports of significant cardiovascular toxicities related to both rofecoxib and celecoxib in several well-publicized sporadic adenoma chemoprevention trials. Because of the risk-benefit balance in our special patient population, namely high risk of malignancy but lower cardiovascular risk due to short trial design and younger patients, the trial was allowed to resume following the addition of more strict exclusion criteria and monitoring measures.

Several limitations were evident in this trial. Because the comparison arm, celecoxib alone, has shown significant activity in FAP, we set the bar rather high insofar as expecting a significant further reduction in adenoma burden by adding DFMO. But in designing the trial we did not feel that use of a placebo control was ethical, and to do so would have still begged the question of whether addition of DFMO really afforded any benefit over and above that seen with celecoxib alone. Because recruitment to FAP trials can be challenging, we did include a number of subjects with a rather minimal polyp burden. The difficulty in measuring response in such patients could have been a source of type II error. Selection of a primary endpoint, polyp counts in a defined but limited field, may have been a poor choice, given alternatives of endpoints that actually used more of the information available in a given endoscopic assessment. Might the wrong agent have been paired with DFMO? In the Meyskens trial of sporadic adenomas, a

16

significant reduction in adenoma recurrence was seen with the combination of sulindac and DFMO. Although that trial employed no sulindac comparison arm, the percent reduction in adenoma recurrence was greater than that observed with any other single agent to date. No head-to-head trial has been conducted or is likely between sulindac and celecoxib. Historically, despite some differences in trial design, the percent reduction in adenomas appears to have been greater with sulindac. Consequently, it may be that combining sulindac with DFMO could achieve a greater synergy than was seen in this trial. Such a trial combining sulindac and DFMO in FAP is late in the planning stages (Al Cohen and Eugene Gerner , personal communication).

Conclusion: What have we learned in this trial? The combination of celecoxib and DFMO, in the doses employed, is well-tolerated by this mainly young patient population. Hearing loss does not seem to be an issue clinically or audiometrically. The efficacy of this combination appears marginal to substantial, depending on how the effect is measured. In the future, careful attention to the means used to measure response may be as or more important than the agent(s) employed. This will be especially the case if a measure of “clinical benefit” is insisted upon, as seems evident from FDA pronouncements. Despite their obvious limitations, pre-clinical animal trials will be a mainstay in the process of developing drug combinations for treatment of FAP, given the extraordinary labors involved in conducting human trials of this kind.

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Table S1. Patient Characteristics by Study SiteAll Site 1 Site 2 Site 3 P-value

Characteristic N (%) N (%) N (%) N (%)All Patients 112 (100) 63 (100) 27 (100) 22 (100)Age - median (min-max) 38 (18-63) 37 (18-61) 43 (21-63) 35 (18-61) 0.89Sex 0.16 Male 60 (54) 29 (46) 16 (59) 15 (68) Female 52 (46) 34 (54) 11 (41) 7 (32)

Race 0.04 White 95 (85) 48 (76) 25 (93) 22 (100) Hispanic 11( 10) 11 (17) 0 (0) 0 (0) Black 1 (1) 1 (2) 0 (0) 0 (0) Other 5 (4) 3 (5) 2 (7) 0 (0)

Basis of FAP Diagnosis <0.001 >100 Polyps 44 (39) 7 (11) 25 (93) 12 (55) >10 polyps and age <40 51 (46) 39 (62) 2 (7) 10 (45) >25 polyps and age > 40 17 (15) 17 (27) 0 (0) 0 (0)Colon vs Rectum <0.001 Colon 46 (41) 41 (65) 0 (0) 5 (23) Rectum Only 66 (59) 22 (35) 27 (100) 17 (77)Patients with Baseline Screen 108 (100) 63 (100) 27 (100) 18 (100)Number of polyps* at baseline screen <0.001 0-1 8 (7) 2 (3) 2 (7) 4 (18) 2-4 19 (17) 6 (10) 8 (30) 5 (23) 5-9 40 (36) 25 (40) 10 (37) 5 (23) 10 or more 41 (37) 30 (48) 7 (26) 4 (18)Number of polyps* at least 2 mm at baseline screen 0.03 0-1 33 (29) 15 (24) 11 (41) 7 (32) 2-4 35 (31) 20 (32) 10 (37) 5 (23) 5-9 30 (27) 19 (30) 5 (19) 6 (27) 10 or more 10 (9) 9 (14) 1 (4) 0 (0)Number of polyps* at least 2 mm at baseline screen for evaluable polyps in evaluable patients**

0.02

0-1 22 (32) 10 (24) 9 (47) 3 (43) 2-4 24 (35) 14 (33) 8 (42) 2 (29) 5-9 18 (26) 14 (33) 2 (11) 2 (29) 10 or more 4 (6) 4 (10) 0 (0) 0 (0)

*Polyps are counted from photographs of the planned landmark sites and do not represent all polyps in the patient.**To be evaluable, patients had to 1) have matching polyp data at baseline and six months as well as 2) have completed at least 80% of the treatment both overall and for the final 60 days.

20

Table S2. Treatment Response by Treatment Arm, Separately by InstitutionCXB+DFMO CXB+PBO

Population N Mean (SE) N Mean (SE)Site 1Polyp Counts from Photos (≥2mm)

ITT All 31 -0.08 (0.12) 32 -0.14 (0.11)ITT Measurable 23 -0.11 (0.17) 30 -0.15 (0.12)Evaluable 21 -0.08 (0.18) 21 -0.09 (0.15)

Polyp Burden from PhotosITT All 31 -0.32 (0.08) 32 -0.32 (0.07)ITT Measurable 23 -0.43 (0.09) 30 -0.34 (0.07)Evaluable 21 -0.41 (0.10) 21 -0.32 (0.08)

Polyp Qualitative Improvement from VideoITT All 31 -0.51 (0.12) 32 -0.33 (0.16)ITT Measurable 19 -0.83 (0.16) 27 -0.39 (0.18)Evaluable 17 -0.94 (0.15) 19 -0.55 (0.21)

Site 3Polyp Counts from Photos (≥2mm)

ITT All 12 -0.16 (0.07) 10 -0.09 (0.12)ITT Measurable 9 -0.22 (0.09) 5 -0.18 (0.24)Evaluable 3 -0.07 (0.07) 4 -0.06 (0.26)

Polyp Burden from PhotosITT All 12 -0.23 (0.07) 10 -0.13 (0.06)ITT Measurable 9 -0.30 (0.07) 5 -0.26 (0.07)Evaluable 3 -0.27 (0.08) 4 -0.22 (0.08)

Polyp Qualitative Improvement from VideoITT All 12 -0.13 (0.12) 10 -0.08 (0.21)ITT Measurable 4 -0.40 (0.35) 5 -0.17 (0.45)Evaluable 1 0.20 (NA) 4 -0.10 (0.57)

Site 2 (Potential Broken Blind)Polyp Counts from Photos (≥2mm)

ITT All 14 -0.10 (0.16) 13 0.39 (0.36)ITT Measurable 13 -0.11 (0.18) 9 0.56 (0.52)Evaluable 11 -0.08 (0.21) 8 0.68 (0.58)

Polyp Burden from PhotosITT All 14 -0.38 (0.09) 13 -0.02 (0.14)ITT Measurable 13 -0.41 (0.09) 9 -0.03 (0.2)Evaluable 11 -0.45 (0.10) 8 0.0 (0.23)

Polyp Qualitative Improvement from VideoITT All 14 -0.76 (0.27) 13 -0.22 (0.25)ITT Measurable 12 -0.88 (0.3) 10 -0.28 (0.33)Evaluable 10 -1.02 (0.33) 8 -0.03 (0.34)

21Enrollment

Randomized (N=112)

Figure 1. CONSORT Diagram

22