1 2 3 Protocol for: 4 5 6 Vitamin D/Calcium Polyp Prevention Study 7 8 9 Principal Investigator: 10 11 John A. Baron. M.D. 12 Geisel School of Medicine at Dartmouth 13 Biostatistics & Epidemiology 14 Evergreen Center, Suite 300 15 46 Centerra Parkway 16 Lebanon, NH 03766 17 603-650-3456 (Phone) 18 603-650-3473 (Fax) 19 [email protected] 20 21 NCI Grant Number: R01 CA098286-01A1 22 23 Project Coordination Center: 24 Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire 25 26 Study Centers: 27 Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire 28 Cleveland Clinic Foundation, Cleveland, Ohio 29 University of Colorado, Denver, Colorado 30 Emory University, Atlanta, Georgia 31 University of Iowa, Iowa City, Iowa 32 University of Minnesota, Minneapolis, Minnesota 33 University of North Carolina, Chapel Hill, North Carolina 34 University of South Carolina, Columbia, South Carolina 35 University of Southern California, Los Angeles, California 36 University of Texas, Houston, Texas 37 University of Puerto Rico/ Puerto Rico Medical Center, San Juan 38 39 Current Date and Protocol version number: 9-11-14 version 20.0 40 41 Previous Date and version number: 5-7-14 version 19.0 42 43 Study agents: Calcium carbonate, Vitamin D3 44 45 IND number: 68,361 46

Downloaded From: https://jamanetwork.com/ on 09/26/2021 47 TABLE OF CONTENTS 48 49 Page 50 1.0 Objectives …………………………………………………………………………………. 5 51 2.0 Background and Rationale ………………………………………………………………… 5 52 3.0 Summary of Study Plan …………………………………………………………………… 13 53 3.1 Overview ………………………………………………………………………… 13 54 3.2 Schema ………………………………………………………………………….. 14 55 4.0 Subject Selection and Enrollment ………………………………………………………… 15 56 4.1 Study Population ………………………………………………………………... 15 57 4.2 Participating Centers ……………………………………………………………. 15 58 4.3 Sources and Methods of Recruitment …………………………………………... 17 59 4.4 Enrollment Procedures …………………………………………………………. 18 60 4.5 Run-in procedures and Randomization Assessment …………………….…….. 19 61 4.6 Method of Subject Identification ………………………………………………. 20 62 4.7 Inclusion Criteria ……………………………………………………………….. 20 63 4.8 Exclusion Criteria ………………………………………………………………. 21 64 5.0 Agent Information and Administration …………………………………………………… 22 65 5.1 Name of Agents and Study Arms ………………… ……………………………. 22 66 5.2 Assignment and Duration of Study Treatment …………………..……………… 22 67 5.3 Dose Selection – Safety and Efficacy ……………………………………..…….. 23 68 5.4 Formulation ……………………………………………………………………… 23 69 5.5 Administration …………………………………………………………………... 23 70 5.6 Side Effects ……………………………………………………………………... 23 71 5.7 Contraindications ……………………………………………………………….. 24 72 5.8 Manufacturer and Supplier ……………………………………………………… 24 73 5.9 Packaging, Labeling and Distribution ………………………………………… 24 74 5.10 Storage ………………………………………………………………………….. 25 75 5.11 Blinding and Unblinding Methods …………………………………………… 25 76 5.12 Adherence/Compliance …………………………………………………………. 25 77 5.13 Agent Accountability ………………………………………………………….. 26 78 5.14 Agent Disposal …………………………………………………………………. 26 79 5.15 Assessment of Blinding Effectiveness ……………………………………….. 26 80 6.0 Post-randomization Follow-up ……………………………………………………………. 26 81 6.1 Interval questionnaires ……………………………………………………………. 27 82 6.2 Schedule of Lab Measurements……………………………………………………. 27 83 6.3 Colonoscopy ………………………………………………………………………. 29 84 6.4 SF-36 Health Survey ……………………………………………………………. 30 85 6.5 End of Treatment Visit ……………………………………………………………. 30 86 6.6 End of Treatment Questionnaire ………………………………………………….. 30 87 6.7 End of Treatment Phase…………………………………………………………… 30 88 6.8 Observational Follow Up ………………………………………………………….. 31 89 7.0 Clinical Procedures – Endoscopy …………………………………………………………. 31 90 7.1 Overview of Procedure …………………………………………………………. 31 91 7.2 Quality Assurance ………………………………………………………………… 32 92 8.0 End-point Determination and Pathology …………………………………………………. 32 93 9.0 Laboratory Measurements – Blood Samples ……………………………………………… 33

v 20.0 9-11-14 2

Downloaded From: https://jamanetwork.com/ on 09/26/2021 94 9.1 Laboratories ……………………………………………………………………… 33 95 9.2 Collection and Handling Procedures …………………………………………….. 34 96 9.3 Methods for Laboratory Procedures …………………………………………….. 34 97 10.0 Safety Monitoring and Reporting Adverse Events ……………………………………… 35 98 10.1 Definition, Monitoring and Follow-up ………………………………………. 35 99 10.2 Adverse Event Information and Severity …………………………………….. 35 100 10.3 Serious Adverse Events ……………………………………………………… 35 101 10.4 Reporting …………………………………………………………………….. 36 102 11.0 Data Management ……………………………………………………………………… 36 103 11.1 Central Database ……………………………………………………………… 36 104 11.2 Website ………………………………………………………………………. 36 105 11.3 Data Entry …………………………………………………………………… 36 106 11.4 Computer Security and Safety……………………………………………….. 37 107 11.5 Archiving ……………………………………………………………………. 37 108 11.6 Reports ………………………………………………………………………. 37 109 12.0 Statistical Methods …………………………………………………………………….. 38 110 12.1 Hypotheses ………………………………………………………………….. 38 111 12.2 Sample Size Justification …………………………………………………… 38 112 12.3 Methods for Randomization and Stratification ………………………………. 40 113 12.4 Outcome Measures ………………………………………………………….. 40 114 12.5 Statistical Analysis ………………………………………………………….. 41 115 12.6 Assumptions …………………………………………………………………. 42 116 12.7 Compliance and Missing Data …………………………………………….…. 42 117 12.8 Interim Analyses ……………………………………………………………… 43 118 12.9 Ancillary Analyses…………………………………………………………….. 43 119 13.0 Ethical and Regulatory Considerations ………………………………………………… 43 120 13.1 Form FDA 1572 ……………………………………………………………… 43 121 13.2 IRB Approval ………………………………………………………………… 43 122 13.3 Informed Consent ………………………………………………………….…. 46 123 13.4 Safety and Data Monitoring Plan ………………………………………….…. 46 124 13.5 Project Coordination Center, Sponsor or FDA Monitoring ………………….. 47 125 13.6 Record Retention ……………………………………………………………. 47 126 13.7 Certificate of Confidentiality …………………………………………………. 47 127 14.0 Ancillary Studies…………………………………………………………………………. 48 128 14.1 Calcium/Vitamin D, Biomarkers, and Colon Polyp Prevention……………… 48 129 14.2 Vitamin D/Calcium Mammography Study…………………………………… 52 130 14.3 Kidney Function with Vitamin D/Calcium…………………………… ..……. 57 131 14.4 Participant Beliefs and Bias Study…………………………………………….. 63 132 14.5 Colds and Flu Study ………………………………………………………….. 65 133 14.6 Dartmouth Polyp Prevention Biospecimen Repository……………………….. 68 134 14.7 Genetic variant influencing response to vitamin D and calcium ……………… 69 135 14.8 Early detection of colorectal neoplasia with ctDNA………...... 69 136 14.9 Metabolomic Study of the Effects of Vitamin D and Calcium 137 Supplementation …………………………………………………………...... 70 138 14.10 Vitamin D3, Calcium & Biomarkers of Inflammation & 139 Gut Barrier Function ………………………………………………………. 70 140 14.11 Mechanisms of obesity-related colorectal carcinogenesis ………………. 71 141 14.12 Sex hormones and Colorectal Neoplasia…………………………………. 71

v 20.0 9-11-14 3

Downloaded From: https://jamanetwork.com/ on 09/26/2021 142 14.13 Interaction of Vitamin D and Calcium with Magnesium and 143 Colorectal Adenoma Recurrence…………………………………………. 72 144 14.14 Identification of prognostic epigenetic markers in biopsy tissue…………. 72 145 14.15 Oncoarray Genotyping……………………………………………………. 72 146 14.16 Data Analyses……………………………………………………….……… 73 147 148 15.0 References ……………………………………………………………………..……. 73 149

v 20.0 9-11-14 4

Downloaded From: https://jamanetwork.com/ on 09/26/2021 150 1.0 Objectives 151 152 There is compelling evidence that most colorectal cancers arise from adenomatous tissue, 153 suggesting that prevention of adenomas would prevent colorectal cancer. Our research group 154 recently documented that calcium supplementation inhibits the development of adenomas in the 155 large bowel, and there is good evidence that vitamin D may also protect against colorectal 156 neoplasia. We will now extend our work by studying the chemopreventive effects of vitamin D 157 on colorectal neoplasia, and by investigating whether calcium in combination with vitamin D is 158 more effective than calcium alone in suppressing colorectal neoplasia. 159 160 This is a randomized, double-blind, placebo-controlled chemoprevention trial, investigating 161 whether supplementation with calcium carbonate and/or vitamin D will reduce the risk of 162 adenomas of the large bowel. Our main hypotheses are: 163 164 1. Supplementation with vitamin D3 (1000 IU/day) reduces the risk of new adenomas in patients 165 with a recent history of these tumors. 166 2. Supplementation with calcium carbonate (1200 mg elemental calcium/day) reduces the risk of 167 new adenomas 168 3. Supplementation with both vitamin D3 (1000 IU/day) and calcium carbonate (1200 mg 169 elemental calcium/day) reduces the risk of new adenomas more than supplementation with 170 calcium carbonate alone. 171 172 Our secondary hypotheses are: 173 174 4. Supplementation with vitamin D3, or with vitamin D3 plus calcium carbonate, reduces the risk 175 of new advanced colorectal lesions (colorectal cancer and adenomas with an estimated 176 diameter of one centimeter or greater, those with tubulovillous or villous histology, or those 177 with advanced dysplasia). 178 5. Supplementation with vitamin D3 will have a greater effect among individuals whose initial 179 serum 25-OH vitamin D levels are less than the overall study median, compared with 180 subjects whose levels are greater than the median. 181 6. The effect of supplementation with vitamin D3 will be modified by polymorphisms at the 3' 182 end of the vitamin D receptor gene. 183 184 In addition to these primary and secondary hypotheses, we will also investigate other 185 benefits and risks associated with calcium and vitamin D supplementation. 186 187 2.0 Background and Rationale 188 189 There is clear evidence that colorectal cancer, the most common visceral cancer in the United 190 States,1 is potentially preventable. Neoplastic polyps (adenomas) of the bowel are recognized 191 pre-malignant lesions and effective endpoints for chemoprevention studies. Randomized 192 intervention studies among patients with adenomas can greatly clarify the effects of intake of 193 specific nutrients on the risk of colorectal neoplasia. Epidemiological and experimental data 194 indicating that both vitamin D and calcium exert anti-neoplastic effects in the large bowel are 195 described below. However, evidence from clinical trials is required to clarify the synergistic and 196 independent chemopreventive effects of these two agents. This study is a multi-centered

v 20.0 9-11-14 5

Downloaded From: https://jamanetwork.com/ on 09/26/2021 197 randomized, double-blind, partial factorial trial of the efficacy of calcium carbonate and vitamin 198 D in preventing the occurrence of neoplastic polyps of the large bowel. 199 200 Vitamin D and Colorectal Carcinogenesis 201 202 Vitamin D and its “Classical” Effects 203 Vitamin D refers to a family of related steroid hormones best known for its important role in 204 maintaining calcium and phosphorus homeostasis, and in regulating bone metabolism.2,3 In its 205 classical actions, vitamin D increases intestinal calcium absorption, decreases serum parathyroid 206 hormone (PTH) levels (thus inhibiting bone resorption) and decreases urinary calcium excretion. 207 Markedly depressed vitamin D status causes osteomalacia, a condition characterized by a 208 deficiency of bone matrix that has severe consequences for bone structure. Mildly depressed 209 vitamin D status seems to predispose to osteoporosis, a condition in which total bone volume is 210 reduced but the bone matrix remains relatively normal.4 211 212 There are two precursors to active vitamin D hormones. Vitamin D3 (cholecalciferol) is 213 synthesized in the skin after exposure to ultraviolet light, and may also be obtained from some 214 dietary sources. A related steroid, vitamin D2 (ergocalciferol), is derived solely from the diet. 215 Since the biological activities of these two compounds are thought to be equivalent, they are 216 often referred to together as “vitamin D,” a convention we follow here. Both compounds are 217 metabolized to the most active steroid, 1,25-(OH)2 vitamin D, through successive hydroxylation 218 reactions: first (25-hydroxylation) in the liver, and second (1α-hydroxylation) in the kidney.2,3 219 Since 25-hydroxylation is not closely regulated, 25-OH vitamin D levels reflect overall vitamin 220 D status from combined dietary and sunlight sources. In contrast, 1α-hydroxylation is tightly 221 regulated to maintain homeostasis of calcium, magnesium and phosphorus. Consequently, serum 222 levels of 1,25-(OH)2 vitamin D do not reflect vitamin D status except during clear conditions of 223 deficiency or excess.5 Both hydroxylation reactions can occur outside the liver and kidney -- for 224 example by activated macrophages or by lymphoma cells -- but these do not affect circulating 225 vitamin D levels in the absence of diseases such as sarcoidosis, other granulomatous diseases, or 226 lymphoma.6-8 227 228 Although 1,25-(OH)2 vitamin D is much more potent than 25-OH vitamin D on a molar basis (by 229 a factor of 100 to 1000), the higher circulating levels of 25-OH vitamin D (nanogram 230 concentrations, rather than picogram) may compensate for its lower potency. Thus 25-OH 231 vitamin D seems to have a direct role in calcium metabolism, for example increasing calcium 232 absorption.7,9,10 233 234 There is relatively little vitamin D in natural foods, and for some humans, sunlight is the 235 predominant source.8,11,12 There is some vitamin D in fatty fish and eggs; the amounts vary 236 depending on the time of year the food was harvested and/or the supplementation status of the 237 animals.8,11 In the U.S., milk and breakfast cereals are fortified, but many other developed 238 countries do not fortify any foods. In practice, even in the U.S., fortification is quite variable. 239 Although toxicity from over-fortification has been reported, the added vitamin D is often less 240 than the stated amount and some “fortified” foods have been found to contain no measurable 241 vitamin D.8,11 242 243

v 20.0 9-11-14 6

Downloaded From: https://jamanetwork.com/ on 09/26/2021 244 The Vitamin D Receptor 245 Like all steroid hormones, vitamin D acts through a nuclear ligand-activated receptor, the 246 vitamin-D receptor (VDR), which operates as a heterodimer with the retinoid X receptor (RXR). 247 Levels of expression of the VDR may have clinical implications: it has been suggested that 248 reduced expression of the VDR in the bowel may explain the decrease in calcium absorption 249 seen with aging.4 Despite the importance of the VDR, rapid actions through a cytoplasmic 250 receptor have also been identified.13 251 252 The VDR contains several polymorphisms. Four of these, near the 3' end of the gene, are in 253 strong linkage disequilibrium with each other: a poly-A microsatellite (3'UTR) and variants 254 identified with the enzymes BsmI (intron 8), Apa I (intron 8), and TaqI (exon 9).14-17 Because of 255 the linkage disequilibrium, in most ethnic groups there are essentially only two VDR 3' 256 haplotypes.14,15,18 These appear to have functional significance, since the polymorphisms have 257 been associated with physiological measurements such as bone density and circulating 1,25- 14,15,17,19-22 258 (OH)2 vitamin D levels. However, these polymorphisms are apparently just markers 259 of another functionally active site that has not yet been identified.15,19 There is also a 260 polymorphic site at the 5' end of the VDR gene, recognized by FokI, in the initiation codon.14,17 261 This polymorphism, in contrast to the other VDR variants, results in an alteration in the 262 expressed amino acid sequence.14 263 264 Vitamin D and Carcinogenesis 265 The expression of the nuclear vitamin D receptor in virtually all tissues of the body (including 266 the bowel mucosa) suggests a role for vitamin D beyond mineral regulation. In particular, recent 267 in-vitro studies show that vitamin D and vitamin D analogues can modulate cellular growth and 268 proliferation. In human cell lines from the colon and other organs, these compounds inhibit 269 proliferation, induce differentiation and promote apoptosis.23-27 The antiproliferative effects of 270 vitamin D are apparent in psoriasis: topical and oral vitamin D has been used to treat this 271 hyperproliferative disorder,28-30 and the Apa I polymorphism in the VDR has been associated 272 with risk of the disease.31 273 274 Clearly, vitamin D signaling can have anti-proliferative effects on the large bowel mucosa, 275 causing, for example, inhibition of proliferation in human rectal mucosal explants32 and in 276 biopsies from patients with ulcerative proctocolitis.33 Vitamin D supplementation normalizes the 277 colonic crypt hyperproliferation of vitamin-D deficient animals,34 and inhibits induction of 278 mucosal ornithine decarboxylase by bile acids35 or the bowel carcinogen DMH.36 The VDR is 279 expressed in neoplastic colorectal tissue,37,38 and expression of the VDR has been correlated with 280 the degree of differentiation of colon carcinoma cell lines.39 High receptor expression has been 281 associated with a good prognosis in colorectal cancer.40 In an intervention trial in humans, rectal 282 mucosal proliferation was found to be inversely related to 25-OH vitamin D levels.41 283 284 Furthermore, various vitamin D hormones can inhibit experimental carcinogenesis. 1,25-(OH)2 42 285 vitamin D3 suppresses the growth of solid tumors derived from human cancer cells, and 286 correction of vitamin D deficiency (together with calcium supplementation) decreased 43 287 experimental bowel carcinogenesis in one rodent model. 1,25-(OH)2 vitamin D and synthetic 288 analogues reduce polyp size and number in Min mice.44 Even in animals that are vitamin-D 289 replete, experimental bowel carcinogenesis has been suppressed by supplementation with 1- 45 46 36,47 290 (OH) vitamin D, vitamin D3 and 1,25-(OH)2 vitamin D. There are some negative data, 291 however.48

v 20.0 9-11-14 7

Downloaded From: https://jamanetwork.com/ on 09/26/2021 292 293 Several molecular mechanisms seem to underlie these effects. Ligand-bound VDR can arrest 294 cells in the G1 stage of the cell cycle, probably through modulation of cell cycle proteins such as 295 cyclin D1.49-51 Vitamin D signaling has important connections to several growth factor 296 pathways. It interferes with epidermal growth factor (EGF) signaling (perhaps by reducing 297 expression of the EGF receptor),49,52,53 reduces expression of the insulin-like growth factor-1 298 receptor,49 and inhibits IGF-1 signaling generally.54 Vitamin D also can reduce expression of c- 299 myc, c-fos and c-jun oncogenes, and suppress telomerase and angiogenesis.49,50,52,55 The 300 apoptotic effects of these hormones may occur through induction of bak and transforming 301 growth factor β or through inhibition of expression of bcl-2.27,49 Vitamin D signaling also seems 302 to play a role in the metabolism (and detoxification) of bile acids in the bowel.56 Finally, 303 vitamin D appears to have important immunological effects, including modulation of T-cell- 304 mediated immunity.6,57,58 305 306 Vitamin D and Human Colorectal Cancer 307 The earliest indications that vitamin D might protect against colorectal carcinogenesis were 308 ecological analyses showing that in the U.S., the risk of colorectal cancer is inversely associated 309 with average sunlight patterns.59,60 More compelling evidence is provided by data regarding 310 dietary vitamin D intake, which has repeatedly been inversely associated with colorectal cancer 311 61-66 and colorectal adenomas,65,67 although there are also studies that do not support an 312 association.68-73 There is likely to be a conservative bias in these studies: inconsistent vitamin D 313 fortification and variable amounts in natural foods create considerable measurement error in 314 dietary assessment.8 Furthermore, the fact that in the U.S. dietary intake of both calcium and 315 vitamin D is largely derived from dairy products makes it difficult to separate the effects of these 316 two nutrients. 317 318 Investigations of supplemental or total vitamin D intake point even more clearly to a protective 319 effect of vitamin D.66,74-79 This is not surprising, since such studies focus on a wider, higher 320 range of intake than dietary studies, and recall of vitamin D supplement use is likely to be more 321 precise than dietary assessment. Investigation of serum vitamin D brings further advantages, 322 since 25-OH vitamin D levels take into account sunlight exposure and are not dependent on 323 dietary recall. 25-OH vitamin D levels have repeatedly been inversely associated with risk of 70,80-83 324 colorectal neoplasia. As discussed above, 1,25-(OH)2 vitamin D levels are tightly 325 regulated and do not reflect vitamin D status; associations with this steroid have not been 326 particularly striking. Interactions of vitamin D with calcium are discussed further below. The 327 association of vitamin D receptor polymorphisms with the risk of colorectal neoplasia has been 328 investigated in a few studies. Overall, the FokI polymorphism seems unrelated to risk,83-85 but 329 one investigation reported an association with large adenomas among subjects with low calcium 330 or vitamin D intake.85 The variant alleles of the 3' polymorphisms have more clearly been 331 related to colon cancer,84,86 particularly among subjects with low calcium or vitamin D intake.86 332 In our Calcium Polyp Prevention Study, however, the VDR variants did not appear to modify the 333 association of vitamin D with adenoma risk. In this study, we will assay TaqI polymorphisms (at 334 the 3’ end of the gene) as well as the (5’) FokI polymorphisms. We hypothesize that the former 335 – but not the latter – will modify the effect of vitamin D treatment. 336 337 The epidemiological data described above suggest that circulating 25-OH vitamin D levels are 338 more closely associated with colorectal cancer risk than are the highly regulated 1,25-(OH)2 339 vitamin D levels. This is a plausible pattern for two reasons. As noted above, the circulating

v 20.0 9-11-14 8

Downloaded From: https://jamanetwork.com/ on 09/26/2021 340 concentrations of 25-OH vitamin D, approximately 3 orders of magnitude higher than those for 341 1,25-(OH)2 vitamin D, seem to be high enough to overcome their relatively low affinity for the 342 VDR. Also, there is emerging evidence that 1α-hydroxylation is constitutively expressed in 343 normal, adenomatous, and malignant colon tissue87-90 (H. Cross, personal communication). Thus 344 high levels of circulating 25-OH vitamin D have the potential to increase intracellular levels of 345 the more active 1,25-(OH)2 form. A similar situation holds for prostate cancer, since human 346 prostate cancer cell lines have been shown to 1α-hydroxylate vitamin D,91 and in these cell lines, 92,93 347 25-OH vitamin D inhibits proliferation with potency similar to 1,25-(OH)2 vitamin D. Thus 348 vitamin D may be effective for prostate cancer chemoprevention even in vitamin D-replete 349 men.91,92 These patterns indicate that doses of vitamin D that are not associated with 350 hypercalcemia may nonetheless have important chemopreventive effects. 351 352 Vitamin D Dose 353 The Institute of Medicine recently increased the recommended daily vitamin D intake for adults 354 to 600 IU per day among those over 50 years old and 800 IU/day for those over 70.94 The 355 NOAEL (no observed adverse effect level) was increased to 4000 IU/day for adults. 94 356 357 No one has investigated the optimal vitamin D dose or serum level for anti-neoplastic effects in 358 humans. From the point of view of bone health, vitamin D levels should be high enough to 359 suppress PTH levels and thus minimize bone resorption. Frank vitamin D deficiency with 360 hyperparathyroidism tends to occur with 25-OH vitamin D levels less than about 30 nmol/L.95 361 However, suppression of PTH does not occur until 25-OH vitamin D levels are above 75-100 362 nmol/L.96-98 These levels become more difficult to sustain among older persons. With age, there 8,11 363 is a decrease in the ability of the skin to produce vitamin D3, a factor that compounds the 364 diminishing periods of time spent outdoors with aging. Moreover, intestinal absorption of 365 vitamin D is impaired in the elderly, and renal 1-hydroxylation of vitamin D declines with 366 age.4,99 367 368 Multivitamins and calcium/vitamin D supplements typically provide 400 IU per day, but 369 numerous intervention studies (reviewed in96,100) show that this dose will not suppress PTH in 370 the overwhelming majority of North American adults. A more effective dose is 1000 IU per day, 371 which will raise 25-OH vitamin D levels toward the desired range, and leave a substantial 372 margin of safety, even after taking into consideration dietary intake. The rate of increase in 25- 373 OH vitamin D levels after daily vitamin D is admittedly variable, but most studies indicate that 374 80-90% of the steady state level is achieved within a few months.101-106 One investigation found 375 that both older and younger subjects responded similarly to a dose of 800 IU of vitamin D3 daily, 376 slightly below the dose we propose.101 377 378 Safety of Vitamin D Supplementation 379 In sufficiently high doses, vitamin D is toxic, largely because of the induction of hypercalcemia, 380 which can lead to renal toxicity (decreased GFR and diminished concentrating capability), 381 metastatic calcification, nausea, vomiting, and CNS effects.8 In doses not associated with 382 hypercalcemia, vitamin D probably has no toxicity. 383 384 We will use vitamin D3 as a supplement, thereby avoiding the toxicity risks associated with 1,25- 385 (OH)2 vitamin D or 25-OH vitamin D. On theoretical grounds, this approach is appealing, since 386 the supplementation would be with a pro-hormone, a choice that takes advantage of natural 387 metabolism to generate the most active moiety. Supplementation with even large doses of

v 20.0 9-11-14 9

Downloaded From: https://jamanetwork.com/ on 09/26/2021 388 vitamin D does not increase total 1,25-(OH)2 vitamin D levels in individuals who are not vitamin 389 D deficient,4 although toxicity may still ensue. 390 391 Vitamin D in doses of 800-1800 IU/day has been found to be safe in several studies (largely 392 investigations of treatment or prevention of osteoporosis), even when given with 1000 mg or 393 more of calcium per day.103,107-113 Several of these studies had treatment periods between 2 and 394 3 years.96,114 Vitamin D doses more than twice as large as that proposed are considered safe8 and 395 carry a large margin of safety. A 1-year pilot study of the regimen which we will use -- 1000 IU 396 of vitamin D administered with 1200 mg of calcium – indicated no toxicity (H. Newmark, 397 personal communication). 398 399 Exposure to sunlight or UV radiation does not in itself lead to vitamin D toxicity, since after a 400 minimal erythema dose, further exposure does not lead to increased 25-OH vitamin D levels; 401 increased degradation counterbalances increased production.8,96 Therefore, even with cutaneous 402 production of vitamin D, the dose we propose will not lead to toxicity among individuals who do 403 not otherwise have disturbances of calcium metabolism. (Individuals with such disturbances 404 will be excluded from the study.) 405 406 Even for the general population, vitamin D supplementation is likely to be beneficial. One study 407 of older Europeans showed that 36% of men and 47% of women had vitamin D deficiency (25- 408 OH vitamin D levels < 30 nmol/L).95 In a younger French population (median age 50 years), 409 14% were found to be vitamin D deficient and 75% had non-suppressed parathyroid hormone 410 (25-OH vitamin D levels below 78 nmol/l).97 Even in sunny countries, low vitamin D levels 411 may be surprisingly common (almost 3/4 of individuals in one study) because of avoidance of 412 direct sun exposure.115,116 413 414 Despite supplementation of milk and breakfast cereals, suboptimal vitamin D status is also 415 common in the U.S. The median intake in young U.S. women has been estimated at 114 IU/day, 416 and only 90 IU/day in older women, both well below recommended levels.8 Among medical 417 inpatients in Boston, 57% were found to have hypovitaminosis D (defined as 25-OH vitamin D 418 levels < 37 nmol/L).117 Even among “healthy” patients under 65 years old (not homebound or 419 chronically ill), 42% were vitamin D deficient, as were 37% of those with estimated vitamin D 420 intakes exceeding the recommended 600 IU/day. Over 90% had 25-OH vitamin D levels below 421 75 nmol/L. Among participants in the Women's Health and Aging Studies (based on a random 422 sample of Medicare beneficiaries), 45% of women with no or minimal disability had 25-OH 423 vitamin D levels at or below 50 nmol/L.118 In our Calcium Polyp Prevention Study, 4.9% of 424 subjects were frankly vitamin D deficient at baseline, and 54% likely had non-suppressed 425 parathyroid hormone (25-OH vitamin D levels < 75 nmol/L). 426 427 Calcium and Colorectal Neoplasia 428 429 Bile Acids, Calcium and Colorectal Carcinogenesis 430 A consistent finding in animal studies is that bile acids and soluble fatty acids in the stool 431 promote colorectal carcinogenesis, probably through irritative/proliferative effects on the bowel 432 mucosa.119-123 Almost twenty years ago it was proposed that calcium in the water phase of the 433 stool could bind and precipitate these carcinogenic factors, thus rendering them relatively 434 inactive and sparing the mucosa from their adverse effects.124,125 Although direct effects of

v 20.0 9-11-14 10

Downloaded From: https://jamanetwork.com/ on 09/26/2021 435 calcium have been proposed,126,127 animal studies have generally supported such an intraluminal, 436 indirect mechanism. Precipitated fat "soaps" have been observed in stool after calcium 437 supplementation, and dietary calcium can ameliorate the hyperproliferative effect of carcinogens, 438 bile acids or small bowel resection on the bowel mucosa.125,128-132 Moreover, in a variety of 439 animal models, calcium intake has protected against experimental bowel carcinogenesis,43,133-136 440 particularly among rats fed a high-fat diet.134,135,137 Some studies suggest a greater effect of 441 calcium on carcinomas than on adenomas,120,135 and on the number of tumors per tumor-bearing 442 animal, rather than on the proportion bearing any tumor.43 443 444 Calcium Intake and Human Colorectal Neoplasia: Observational Data 445 In contrast to the animal data, human epidemiological studies regarding dietary calcium intake 446 and colorectal cancer have yielded mixed findings. Some cohort studies found suggestions of an 447 inverse association of dietary or total calcium intake with colorectal cancer risk,74-77,138 but 448 several have been negative.139-142 Case-control studies have reported similarly inconsistent 449 results,141,142 with findings ranging from a marked inverse association (e.g.,143) to suggestions of 450 an increased risk with high dietary intake.63 Epidemiological investigation of colorectal 451 adenomas has also been conflicting,69,71,72,75,144-148 as have studies regarding the effects of 452 calcium supplements on colorectal neoplasia.77,140,149-152 453 454 Clinical Trials of Calcium and Colorectal Carcinogenesis 455 The strongest support for a protective effect of calcium intake on colorectal neoplasia has come 456 from randomized chemoprevention trials, which avoid many of the difficulties of observational 457 investigation. In a clinical trial conducted by our research group, we studied the effect of 4 years 458 of supplementation with 1200 mg daily of elemental calcium among patients with a recent 459 history of colorectal adenomas. Calcium treatment reduced the risk of any recurrent adenoma by 460 17% (95% CI 2-33%), and reduced the numbers of recurrent adenomas by 25% (95% CI 4-40%). 461 There was a more pronounced effect on lesions with advanced features (54% risk reduction, 95% 462 CI 17-74%). Very similar findings were reported from a smaller European trial153 in which 463 calcium supplementation (2 gm/day) conferred a 25% (95% CI -29% to 57%) reduction in risk. 464 465 Two other, smaller, clinical trials also used calcium, though in quite different contexts. In a 466 study among patients with FAP, calcium supplementation (600 mg/day) provided no clear 467 benefit with regard to polyp counts, although the treated group did have a smaller increase in 468 polyp number than the placebo group.154 Another trial of 116 patients who had small (<10 mm) 469 polyps left in place, tested a mixture of antioxidants (selenium, beta carotene, alpha tocopherol 470 and ascorbic acid) and calcium (1.6 gm/day). The mixture had no effect on the growth of 471 existing polyps, but significantly reduced the risk of new adenomas (risk ratio = 0.70).155 472 473 Investigations of the effect of calcium supplementation on proliferative indices have been 474 inconsistent; some studies reported no beneficial effect,156-162 but others found a reduction in 475 proliferation.163-168 Trials of other biological markers have also been inconsistent. In some 476 investigations, anti-carcinogenic effects were observed: a reduction in the cytotoxicity of fecal 477 water,160,169,170 a reduced proportion of secondary bile acids in the bile acid pool,160,170,171 and 478 lowered bile acid concentration in fecal water.171 However, other investigations reported 479 contrary findings: no change,157,169 or an increase156,159,172 in the concentration of bile acids in 480 the water phase of stool. 481 482 Interactions of Vitamin D and Calcium

v 20.0 9-11-14 11

Downloaded From: https://jamanetwork.com/ on 09/26/2021 483 Vitamin D and calcium are intertwined physiologically, and an important research goal is 484 understanding their independent and synergistic effects. One experimental study in rodents 485 found that calcium and vitamin D3 supplementation together had a smaller protective effect than 486 either supplement alone.134 However, most studies have reported that vitamin D 487 supplementation has a stronger anti-neoplastic effect in animals given relatively high-calcium 488 diets.43,45,46 Because of the measurement error associated with these two exposures, it is to be 489 expected that human observational studies may have difficulty clarifying any interactive effects. 490 Indeed several studies seem to suggest that the effects of dietary calcium and vitamin D are 491 independent of each other.64,69,74,77 However, in two large cohort studies with careful dietary 492 assessment,74,138 there was clear evidence of a positive interaction between the two nutrients. 493 Also, in our Calcium Polyp Prevention Study (see Preliminary Studies, below), there were strong 494 indications that vitamin D enhanced the chemopreventive effect of calcium. Among subjects 495 whose baseline 25-OH vitamin D levels were below the median, calcium had no effect on risk of 496 one or more adenomas after randomization (adjusted risk ratio 1.06; 95% CI 0.82-1.22). In 497 contrast, among subjects with 25-OH vitamin D levels above the median, calcium substantially 498 lowered the risk of adenoma occurrence (adjusted risk ratio for calcium 0.71; 95% CI 0.57-0.88; 499 p for interaction = 0.01). As expected, the calcium effect was similar in subjects with higher and 500 lower levels of 1,25-(OH)2 vitamin D. These important data suggest that vitamin D and calcium 501 may work together to reduce the risk of colorectal neoplasia. 502 503 Calcium Chemoprevention 504 The positive clinical trial findings regarding calcium chemoprevention of large bowel neoplasia 505 are exciting and important from both scientific and public health points of view. They help 506 clarify contradictory observational studies, provide clues to the etiology of colorectal neoplasia, 507 and advance the identification of effective chemopreventive strategies. 508 509 Calcium supplementation is widely used and is safe. The Institute of Medicine’s recommended 510 calcium intake for adults 19 through 50 years old is 1000 mg/day, for adults 51 through 70 years 511 it is1200 mg/day for women and 1000 mg/day for men, and for those over 70 years it is 1200 512 mg/day.94 Intake up to 2000 mg/day in adults over 50 is considered safe.94 The main potential 513 toxicities of calcium supplementation are urinary stones and hypercalcemia (which can lead to 514 renal insufficiency and alkalosis, the “milk-alkali syndrome”).8,94,173,174 We will exclude from 515 our study all subjects with renal insufficiency or a history of kidney stones. Milk alkali 516 syndrome is rare in healthy people,8,94 and has not been noted in trials of calcium and vitamin D 517 supplementation that used doses similar to those we propose. 518 519 Summary 520 Extensive experimental and observational data suggest that intake of both calcium and vitamin D 521 exert protective effects on colorectal neoplasia. Building on our previous work, we will now 522 investigate the chemopreventive effect of vitamin D in the large bowel, to study whether both 523 agents together are more effective than calcium alone, and to confirm our positive finding 524 regarding calcium. Our goal is the development of chemopreventive combinations that will 525 reduce risk of colorectal neoplasia sufficiently to permit the lengthening of surveillance intervals 526 in most patients and to clarify important issues regarding the mechanisms of colorectal 527 carcinogenesis and chemoprevention. 528 529 530 3.0 Summary of Study Plan

v 20.0 9-11-14 12

Downloaded From: https://jamanetwork.com/ on 09/26/2021 531 532 3.1 Overview 533 This study is a double-blind, placebo-controlled trial of vitamin D and/or calcium 534 supplementation for the prevention of large bowel adenomas. Subjects will be recruited from 11 535 Study Centers in North America. Eligible subjects will have had at least one large bowel 536 adenoma removed in the 4 months prior to study entry and no remaining polyps in the bowel 537 after complete colonoscopic examination. Participants will be randomized in a modified 2 x 2 538 factorial design to vitamin D (1000 IU/day), calcium carbonate (1200 mg elemental 539 calcium/day), both agents, or placebo only. Women who decline to forego calcium 540 supplementation will be randomized only to calcium alone or to calcium plus vitamin D. 541 Randomization will be stratified by gender, Study Center of recruitment, and anticipated follow- 542 up interval (see below), and will be conducted separately for female subjects randomized only to 543 vitamin D. Participants will agree to avoid taking study agents outside the trial and will initially 544 be observed in a 3-month placebo run-in period to identify (and exclude before randomization) 545 subjects likely not to adhere to the study regimen. We originally anticipated enrolling about 546 2500 participants to reach a total of approximately 2000 randomized subjects. Subsequently 547 these numbers were increased to up to 3000 enrolled and up to 2400 randomized to increase 548 representation of minorities, women and high risk subjects. As safety measures, blood levels of 549 calcium, creatinine and 25-(OH)-vitamin D will be obtained at baseline and 1 year after 550 randomization, as well as 3 years after randomization for subjects with a 5-year surveillance 551 cycle. Every six months after randomization subjects will complete a questionnaire regarding 552 compliance with study agents, use of medications and vitamin/mineral supplements, illnesses, 553 hospitalizations, and dietary intake of calcium and vitamin D. 554 555 The endpoint of the study will be new adenomas detected on follow-up colonoscopy. 556 These examinations are scheduled to occur either 3 years or 5 years after the qualifying 557 examination, depending on the follow-up interval recommended by each patient's endoscopist. 558 Some patients may, for medical reasons, have a colonoscopy at a time other than 3 or 5 years 559 after the qualifying examination. Information from these exams will be included in analyses 560 where appropriate. In the primary analyses, the occurrence of new neoplastic polyps in the 561 interval between randomization and the follow-up exam will be compared between subjects 562 randomized to vitamin D (with or without calcium) versus those randomized to placebo (with or 563 without calcium), between subjects randomized to calcium (with or without vitamin D) versus 564 those randomized to placebo (with or without vitamin D, excluding women electing to receive 565 calcium and therefore cannot participate in the calcium component of the study), and between 566 those randomized to calcium plus vitamin D versus those randomized to calcium alone. In 567 secondary analyses, we will examine the effect of calcium plus vitamin D versus vitamin D 568 alone, and the impact of baseline vitamin D levels and VDR polymorphisms on the vitamin D 569 effects. Effects on advanced adenomas will also be assessed. 570

v 20.0 9-11-14 13

Downloaded From: https://jamanetwork.com/ on 09/26/2021 571 3.2 Schema 572 573 Colorectal Chemoprevention with Calcium and Vitamin D 574 John A. Baron, Dartmouth Medical School 575 576 577 Men and women with a recent colonoscopy in which an adenoma was removed, 578 with acceptable baseline measurements of calcium, creatinine and 25-(OH)-vitamin D 579 580 581 582 583 Three month RUN-IN period (about 3000 participants) 584 585 586 587 588 Randomize about 2400 men and women to: 589 placebo (360 men and 84 women) or 590 1200 mg elemental calcium (360 men and 84 women) or 591 1000 IU vitamin D (360 men and 84 women) or 592 both agents (360 men and 84 women) daily 593 -OR- 594 Randomize women who want to take calcium to: 595 1200 mg elemental calcium (312 women) or 596 1200 mg elemental calcium and 1000 IU vitamin D (312 women) daily 597 598 599 600 601 Subject questionnaires at 6-month intervals 602 603 604 605 606 Measurements of calcium, creatinine and/or 25-(OH)-vitamin D at year 1, year 3 and, if 607 applicable, year 5 608 609 610 611 612 Follow-up colonoscopy at 3 or 5 years 613 614 615 616 617 Number and type of new colorectal adenomas found 618

v 20.0 9-11-14 14

Downloaded From: https://jamanetwork.com/ on 09/26/2021 619 4.0 Subject Selection and Enrollment 620 621 4.1 Study Population 622 Study subjects will be healthy volunteers who have received preventive care from a 623 gastroenterology clinic. They will be recruited to the study because they are at risk for future 624 adenomas based on the occurrence of at least one previous adenoma removed at a surveillance 625 colonoscopy (qualifying colonoscopy) occurring within the previous four months. Recruited 626 subjects will be between 45 and 75 years of age at enrollment. Current preventive health care 627 guidelines suggest an initial screening colonoscopy at 50 years of age. Individuals younger than 628 45 are excluded because adenomas occurring in that age group may be due to the presence of a 629 familial colon cancer syndrome. Thus, they are likely to represent a different population with a 630 different mechanism. The upper age limit for inclusion in the trial is used to increase the 631 likelihood of successful compliance and completion of this long-term chemoprevention trial. 632 633 Individuals of both genders and all racial and ethnic populations will be encouraged to 634 participate in the trial. Based on previous trials, we anticipate that approximately 60% of 635 enrolled subjects will be males and 40% females. This accords with the demographics of 636 adenoma prevalence, which is somewhat higher among men. We expect the ethnic and racial 637 make-up of the subject population to approximate that of the U.S. population. The diversity of 638 populations in the catchment areas of our 11 Study Centers makes this a realistic goal. 639 640 4.2 Participating Study Centers and Recruitment Sites 641 Subjects will be recruited from the gastrointestinal and surgical services of the eleven 642 participating Study Centers. Principal Investigators (PI) for each Study Center are provided 643 below. Recruitment or referral will also occur from ancillary sites, including hospitals, clinics, 644 cancer centers, university medical practices, Veterans Affairs Medical Centers, health 645 maintenance organizations, and private gastroenterology practices associated with these centers, 646 as listed below. Those recruitment sites listed below that have an Institutional Review Board 647 (IRB) that provides human subject’s oversight for this study that is separate from that of the 648 Study Center are indicated with an asterisk. 649 650 1. Dartmouth Hitchcock Medical Center, Lebanon, NH 651 (PI: Richard Rothstein, MD, then Douglas Robertson, MD) 652 a. Dartmouth-Hitchcock Manchester, Manchester, NH 653 b. Dartmouth-Hitchcock Keene, Keene, NH 654 c. Portsmouth Gastroenterology, Portsmouth, NH* 655 d. Portland Gastroenterology Association, Portland, ME* 656 e. Concord Gastroenterology, PA, Concord, NH* 657 f. VA Medical Center, White River Junction, VT 658 2. Cleveland Clinic Foundation, Cleveland, OH 659 (PI: Gerald Beck, PhD) 660 a. Cleveland Clinic- Beachwood Family Health Center, Cleveland, OH 661 b. Cleveland Clinic-Independence Family Health Center, Independence, 662 OH 663 c. Cleveland Clinic-Lorain Family Health Center, Lorain, OH 664 d. Cleveland Clinic-Strongsville Family Health Center, Strongsville, OH 665 e. Cleveland Clinic-Chagrin Falls Family Health Center, Chagrin Falls, 666 OH

v 20.0 9-11-14 15

Downloaded From: https://jamanetwork.com/ on 09/26/2021 667 f. Cleveland Clinic-Elyria Family Health Center, Elyria, OH 668 g. Cleveland Clinic-Lakewood Family Health Center, Lakewood, OH 669 h. Cleveland Clinic-Solon Family Health Center, Solon, OH 670 i. Cleveland Clinic-Westlake Family Health Center, Westlake, OH 671 j. Cleveland Clinic-Willoughby Hills Family Health Center, Willoughby 672 Hills, OH 673 k. Metrohealth Medical Center, Cleveland, OH* 674 l. Cleveland Clinic – Wooster Family Health Center, Wooster, OH 675 3. University of Colorado, Denver, CO 676 (PI: Dennis Ahnen, MD) 677 a. Rocky Mountain Gastroenterology, Aurora, CO 678 b. Rocky Mountain Gastroenterology, Lakewood, CO 679 c. South Denver Gastroenterology, Denver, CO 680 d. University Hospital, Aurora, CO 681 e. Arapahoe Gastroenterology, Denver, CO 682 f. Arapahoe Gastroenterology, Littleton, CO 683 g. VA Medical Center, Denver, CO 684 4. Emory University, Atlanta, GA 685 (PI: Jack S. Mandel, PhD, then Michael Goodman, MD, MPH) 686 a. VA Medical Center, Decatur, GA* 687 b. Consultative Gastroenterology, Atlanta, GA 688 5. University of Iowa, Iowa City, IA 689 (PI: Robert W. Summers, MD) 690 a. Gastroenterologists, P.C.I, Cedar Rapids, IA 691 b. Internists, Cedar Rapids, IA 692 c. Gastroenterology Assoc. of Iowa City, Iowa City, IA 693 d. Cedar Valley Medical Specialists, Waterloo, IA 694 e. Dubuque Internal Medicine, Dubuque, IA 695 f. Iowa Clinic, Gastroenterology, Des Moines, IA 696 g. Iowa Digestive Disease Center, PC, Des Moines, IA 697 6. University of Minnesota, Minneapolis, MN 698 (PI: Tim Church, PhD) 699 a. Minnesota Gastroenterology, Minneapolis, MN 700 b. VA Medical Center, Minneapolis, MN* 701 7. University of North Carolina, Chapel Hill, NC 702 (PI: Robert S. Sandler, MD) 703 a. Cary Gastroenterology Association, Cary, NC 704 b. Regional Gastroenterology Association, Durham, NC 705 8. University of South Carolina, Columbia, SC 706 (PIs: Roberd M. Bostick, MD, March E. Seabrook, MD) 707 a. Consultants in Gastroenterology, West Columbia, SC 708 9. University of Southern California, Los Angeles, CA 709 (PI: Robert Haile, PhD, then Jane Figueiredo, PhD) 710 a. Los Angeles County Hospital, Los Angeles, CA 711 b. South Bay Gastroenterology Medical Group 712 c. Kaiser/Sunset, Los Angeles, CA* 713 10. University of Texas, Houston, TX 714 (PI: Robert Bresalier, MD)

v 20.0 9-11-14 16

Downloaded From: https://jamanetwork.com/ on 09/26/2021 715 a. MD Anderson Cancer Center, Houston, TX 716 b. Medical Center Endoscopy, Houston, TX 717 11. University of Puerto Rico, San Juan, PR 718 (PI: Marcia R. Cruz-Correa, MD, PhD) 719 a. VA Caribbean Healthcare System, San Juan, PR* 720 b. Active GI Group, San Juan, PR 721 c. Instituto de Gastroenterología, San Juan, PR 722 d. Hato Rey Medical Gastroenterology, Hato Rey, PR 723 e. Grupo Gastroenterologico del Este, Humacao PR 724 725 4.3 Sources and Methods of Recruitment 726 Study coordinators or treating medical staff will identify potential subjects through review of 727 colonoscopy schedules and pathology reports at the participating centers and associated 728 practices. All potential subjects must have had at least one adenoma that is at least 2 mm in size 729 removed from the large bowel in the four months before entry into the study. For these 730 diagnoses, we will accept the readings of the local pathologists. For individuals who meet this 731 initial criterion, a preliminary determination of eligibility will then be made through review of 732 available medical records and contact with the participating physicians. Those individuals who 733 continue to be eligible following this review will be assigned a study ID number for tracking and 734 reporting purposes upon submission of preliminary eligibility data to the Dartmouth Project 735 Coordination Center. This eligibility data will include the individual’s date of birth and the date 736 of the qualifying colonoscopy exam (components of a limited data set as defined by HIPAA) as 737 permitted by local regulatory requirements. 738 739 Study Centers will implement the following process as permitted/required by local IRBs: 740 Potential subjects will be mailed a packet of information about the study, preferably from their 741 personal physician or clinical staff with whom they are familiar. This packet (packet #1) will 742 include: 743 744 • Introductory letter with a short description of the study and the name of the study coordinator 745 who will be contacting the patient to determine their interest in participation. 746 • Post card that may be returned to decline further contact regarding the study or to provide 747 information about a good time to reach them by phone 748 • Study brochure describing the study in more detail 749 • Medical release authorization (may be required to verify eligibility) 750 751 The study coordinator will phone those individuals who do not decline further contact within 14 752 days to discuss their interest in the study, to confirm eligibility and to set up an intake 753 appointment for those individuals who are interested in study participation. A second 754 information packet (packet #2) will be mailed to these individuals containing: 755 756 • Letter confirming the intake appointment date and time 757 • Checklist of what to bring to the intake appointment 758 • Block Food Frequency questionnaire 759 • RAND SF-36 Health Survey 760 • Copy of the study’s Informed Consent document

v 20.0 9-11-14 17

Downloaded From: https://jamanetwork.com/ on 09/26/2021 761 • Brochure from the NIH/NCI called “Taking Part in Clinical Trials: Cancer Prevention 762 Studies – What Participants Need to Know “ or brochure from The Office for Human 763 Research Protections called “Becoming a Research Volunteer: It's Your Decision” 764 765 4.4 Enrollment Procedures 766 An intake appointment will be scheduled within 120 days of the qualifying colonoscopy for 767 those individuals who continue to be interested in study participation. At the intake 768 appointment, an informed consent discussion will take place between the prospective participant 769 and the study coordinator. After obtaining signed consent (the date of the signed consent is the 770 Enrollment Date for the participant), the study coordinator will confirm eligibility and document 771 the subject’s general demographic information, general health and medical history including 772 previous colorectal procedures and adenomas, use of nutritional supplements and medications, 773 recent sun exposure and exercise, smoking history, and family history of cancer or adenomas 774 (the Intake Questionnaire). The responses to these questions will be entered by the study 775 coordinator into a secure web-based form that transmits data directly to the Dartmouth Project 776 Coordination Center in real-time over the Internet. The participant will also be asked to 777 complete and submit to the study coordinator a Block Food Frequency Questionnaire and an SF- 778 36 Health Survey. The Block Food Frequency Questionnaire is a validated self-administered 779 questionnaire that is intended for epidemiologic and clinical use and captures an individual’s 780 usual dietary food intake. The SF-36 Health Survey is a validated 36-item self-administered 781 questionnaire on health status that assesses eight different health concepts and includes 782 perceptions of both physical and mental health. 783 784 In a final component of the intake appointment, the study coordinator will measure and 785 document the participant’s current height and weight, and obtain the baseline blood draw. 786 Whenever possible, this should be a fasting blood draw (the participant should not eat food or 787 drink beverages other than water for 8 hours prior to the blood draw). Regardless, the time of 788 the last food or beverage intake will be collected. Pre-packaged bar-coded blood collection kits 789 will be provided for each participant. Standard and detailed operating procedures will be 790 provided by the Dartmouth Project Coordination Center for adequate and safe collection, 791 processing, storage and shipment of these biospecimens. 792 793 Upon completion of the intake appointment, all participants will be given a study packet (packet 794 #3) containing: 795 796 • Copy of the signed and dated informed consent form 797 • Participant Information Booklet 798 • Phone card -optional (Additional phone cards will be sent or given to subjects from the Study 799 Center on a yearly basis.) 800 • 7-day pill dispenser 801 • Bottle of run-in study pills 802 • Study Identification Card 803 • Bottle of multivitamins without any vitamin D or calcium -optional per subject request 804 (Additional bottles will be sent or given to subjects from the Study Center as needed.) 805 • Participant Drug Information Card (optional per local IRB requirement) 806 807

v 20.0 9-11-14 18

Downloaded From: https://jamanetwork.com/ on 09/26/2021 808 The study coordinator will ask the participant to take the first study pill. The coordinator will 809 document the taking of the first pill at the intake appointment or ask the participant to confirm by 810 postcard or telephone if taken later. All prospective participants who complete the intake 811 appointment will be reimbursed $100 to defray the time and expense of the 812 recruitment/enrollment activities. 813 814 The study coordinator will ship blood samples for Vitamin D measurements to the UCLA 815 Nutrition Research Center at the next scheduled shipment date. (The UCLA laboratory will 816 provide the results of these measurements directly to the Project Coordination Center.) In 817 addition, within 15 days after the intake appointment, the study coordinator will submit to the 818 Dartmouth Project Coordination Center the qualifying pathology and endoscopy reports and the 819 results of the local lab measurements of calcium and creatinine. These lab results and clinical 820 reports will be required to confirm the eligibility of the participant prior to randomization. If the 821 results for calcium or creatinine are outside of the range for eligibility (see section 4.7 below) 822 then the subject and their health care provider will be notified of the result in writing. If the 823 result for 25-(OH)- vitamin D is outside of the range for eligibility (see section 4.7 below), then 824 the subject and their health care provider will be notified in writing whether the result is “too 825 high” or “too low.” The exact measurement will not be provided because these study 826 measurements are not performed in a CLIA certified lab and so can be used for research 827 purposes only. The health care providers for the affected subjects will be advised to obtain a 25- 828 (OH)- vitamin D level at a CLIA-approved laboratory if further assessment is thought to be 829 indicated. 830 831 Other information to be sent to the Project Coordination Center includes the completed Food 832 Frequency Questionnaire, which will be mailed, and the SF-36 Health Survey, which will be 833 mailed until the web form is available for data entry and transmission over the internet. These 834 must be received by the Project Coordination Center within 60 days of the intake appointment. 835 Finally, with the consent of the participant, the coordinator will also ship (within 60 days of the 836 intake appointment) blood samples to the Clinical & Molecular Pharmacology Shared Resource 837 of the Norris Cotton Cancer Center to be stored for future research on colorectal neoplasia and 838 other disorders common in the study population. 839 840 Quality Control. The Dartmouth Project Coordination Center will review the pathology and 841 endoscopy reports to confirm the Study Center’s assessment of eligibility before the prospective 842 participant is randomized. 843 844 4.5 Run-in period and randomization assessment 845 At the intake appointment, or immediately upon taking the first run-in pill, the participants will 846 enter an approximately three month long run-in period during which adherence to study agents 847 will be assessed. Approximately 10 weeks after the intake appointment, the study coordinator 848 will contact participants by phone to determine and document continued interest in the study, 849 any change in eligibility or exclusionary criteria, and run-in pill taking compliance. Subjects 850 will be asked to count the number of pills left in their pill bottles and report this number to their 851 study coordinator who will enter it into a web form that will be used to calculate compliance. 852 Participants whose pill counts indicate that they have taken less than 80% of their study pills will 853 not be randomized. Participants will also be given their first ‘interval’ questionnaire on this 854 phone call (see section 6.1 below). This phone call must occur between 56 and 84 days of the 855 intake appointment. After this phone call, participants who continue to be eligible for and

v 20.0 9-11-14 19

Downloaded From: https://jamanetwork.com/ on 09/26/2021 856 interested in study participation will be sent a new supply of study pills by mail from the 857 Dartmouth Project Coordination Center. The new supply will be the randomly assigned study 858 agent. Participants will be provided a postcard or telephone number to report the date the first 859 pill was taken from this shipment. It should be taken within 98 days of the intake appointment. 860 Study coordinators will follow-up to collect this date if the patient forgets to report it. 861 862 4.6 Method of Subject Identification 863 All potentially eligible individuals identified by a review of colonoscopy and pathology records 864 will be assigned a study identification number prior to initial patient contact. The ID number 865 will be in the form SC - RS - ##### . The “SC” is a number ranging from 01-99 and identifies 866 the Study Center from which the subject was recruited. The “RS” is a number ranging from 01- 867 99 and corresponds to the specific recruitment site. The “#####” ranges from 00001-99999 and 868 identifies the individual subject. This number is generated using a modulo 11 method that 869 prevents the occurrence of sequential numbers in order to reduce errors from typing mistakes. If 870 an individual is subsequently enrolled in the study then this same ID number will be used 871 throughout the study. In addition, upon enrollment the subject will also be assigned a two letter 872 study identification code consisting of the first initial of the subject’s first name followed by the 873 last letter of the subject’s last name. This code will be used along with the subject’s study ID 874 number in communications between the Dartmouth Project Coordination Center and the Study 875 Center coordinators. 876 877 4.7 Inclusion criteria 878 1) One or more histologically verified neoplastic polyp (adenoma) that is at least 2 mm in 879 size removed from the large bowel with the entire large bowel examined by 880 colonoscopy and documented to be free of further polyps or areas suspicious for 881 neoplasia within 120 days of study entry 882 2) Anticipated colonoscopic follow-up three years or five years after the qualifying 883 colonoscopy 884 3) Age between 45 and 75 years at enrollment 885 4) (Women)Agreement to avoid pregnancy (i.e. use of standard contraception) 886 5) Willingness to forego calcium supplementation (including multivitamins containing 887 calcium) or, for women only, option of taking calcium supplementation of 1200 888 mg/daily (contained in the study pills) 889 6) Willingness to forego vitamin D supplementation (including multivitamins containing 890 vitamin D) 891 7) Agreement to daily dietary intake of the equivalent of not more than 1200 mg calcium 892 8) Agreement to daily dietary intake of the equivalent of not more than 400 IU vitamin D 893 9) Blood calcium level within normal range 894 10) Blood creatinine level not to exceed 20% above upper limit of normal 895 11) Serum 25-(OH)-vitamin D within lower limit of 12 ng/ml to upper limit of 90 ng/ml 896 12) Ability and willingness to follow the study protocol, as indicated by provision of 897 informed consent to participate 898 13) Good general health, with no severely debilitating diseases or active malignancy that 899 might compromise the patient's ability to complete the study 900 901 4.8 Exclusion criteria 902 (General exclusionary criteria:) 903 1) Participation in another colorectal (bowel) study (intervention study) in the past 5 years

v 20.0 9-11-14 20

Downloaded From: https://jamanetwork.com/ on 09/26/2021 904 2) Current participation in any other clinical trial (intervention study) 905 3) Pregnancy or lactation 906 4) A diagnosis of narcotic or alcohol dependence in the past 5 years 907 5) A diagnosis of dementia (e.g. Alzheimers) in the past 5 years 908 6) A diagnosis of a significant psychiatric disability (e.g. Schizophrenia, refractory bipolar 909 disorder, current severe depression) in the past 5 years 910 911 (Exclusions due to derangement of calcium metabolism or indications/contraindications to 912 study agents) 913 7) Any diagnosis of kidney stones 914 8) A diagnosis of granulomatous diseases, e.g. sarcoidosis, active chronic fungal or 915 mycobacterial infections (tuberculosis, histoplasmosis, coccidioidomycosis, 916 blastomycosis), Berylliosis, Wegener’s granulomatosis in the past 5 years 917 9) A diagnosis of hyperparathyroidism or other serious disturbance of calcium metabolism in 918 the past 5 years 919 10) A diagnosis of severe kidney disease, e.g. chronic renal failure in the past 5 years 920 11) A diagnosis of unexplained hypercalcemia in the past 5 years 921 12) Any diagnosis of osteoporosis with physician recommendation for treatment of low bone 922 mass 923 13) A diagnosis of two or more low trauma fractures in the past 5 years 924 14) A diagnosis of a medical condition requiring treatment with vitamin D (e.g. osteomalacia) 925 in the past 5 years 926 927 (Exclusions due to intestinal or bowel problems:) 928 15) Any diagnosis of invasive carcinoma of the large bowel (even if confined to a polyp) 929 16) Any diagnosis of familial colorectal cancer syndromes, e.g. Familial Adenomatous 930 Polyposis (FAP) (including Gardner syndrome, Turcot’s syndrome), Hereditary 931 Nonpolyposis Colorectal Cancer (HNPCC), Hamartomatous Polyposis syndromes 932 (including Peutz-Jeghers or Familial Juvenile Polyposis) 933 17) Any diagnosis of inflammatory bowel disease, e.g. Crohn’s Disease, Ulcerative Colitis 934 18) A diagnosis of chronic intestinal malabsorption syndromes, e.g. celiac sprue, bacterial 935 overgrowth, chronic pancreatitis, pancreatic insufficiency in the past 5 years 936 19) Any large bowel resection 937 938 (Exclusions due to poor health:) 939 20) A diagnosis of malignancy, other than non-melanoma skin cancer in the past 5 years 940 21) A diagnosis of severe lung disease – class 3 or 4 (e.g. COPD or emphysema requiring 941 oxygen) in the past 5 years 942 22) A diagnosis of severe heart disease: Cardiovascular disease functional class 3 or 4 in the 943 past 5 years 944 23) Any diagnosis of severe liver disease, e.g. Cirrhosis 945 946 947 (Exclusions due to shipping regulations:) 948 24) Any current/past HIV positive diagnosis 949 25) Active hepatitis B, defined as : Hep B surface antigen positive 950 Active hepatitis C, defined as : measurable HCV RNA 951

v 20.0 9-11-14 21

Downloaded From: https://jamanetwork.com/ on 09/26/2021 952 (Drug exclusions:) 953 26) Use of chronic oral cortico-steroid therapy in the past 5 years 954 27) Use of Lithium in the past 5 years 955 28) Use of Phenytoins in the past 5 years 956 29) Use of Quinidine in the past 5 years 957 30) Use of therapeutic vitamin D in the past 5 years 958 959 5.0 Agent Information and Administration 960 961 5.1 Name of agents and study arms 962 963 There will be four arms in the study: 964 1. placebo 965 2. calcium carbonate (3 gm/daily; 1200 mg elemental calcium/daily) 966 3. vitamin D3 (1000 IU/daily) 967 4. calcium carbonate and vitamin D3 968 969 5.2 Assignment and Duration of Study Treatment 970 Men will be randomly assigned to each of the treatment groups at equal frequency. Due to 971 recommendations for high calcium intake among post-menopausal women and the common use 972 of calcium supplements in that population group, women will have the option of being 973 randomized to calcium or to calcium plus vitamin D (arm 2 or 4). In this way, they will receive 974 3 gm of supplemental calcium carbonate daily as part of the study. Based on our experience in 975 the Aspirin-Folate Polyp Prevention Study, we assume that 35% of women will accept the full 976 factorial randomization and 65% will wish to continue calcium supplementation and accept the 977 restricted randomization (two arm). There will be separate randomization procedures for 978 subjects being randomized to both agents and for women taking calcium who will be randomized 979 only to vitamin D. Although it will not be encouraged, at any time after enrollment, women who 980 are in the Full Factorial component of the study will have the option to change their mind and 981 switch to the 2 Arm component of the study. Study tablets shipped to these subjects will be 982 changed accordingly. For statistical analyses purposes they will be analyzed as per the original 983 treatment group that they were randomized to. All randomization will be stratified by Study 984 Center of recruitment and scheduled colonoscopic follow-up (3 years or 5 years). 985 986 The follow-up interval for colonoscopy recommended by a patient's endoscopist, either 3 or 5 987 years, will determine the duration of exposure to the study agents. For a 3 year follow-up 988 interval, the duration of exposure to the study agents will be approximately two and one half 989 years (30 months) due to the subtraction of the initial time required for subject identification and 990 recruitment (up to four months) and a subsequent three month run-in period during which 991 subjects take placebo pills (full factorial) or calcium alone (two arm). Likewise, the duration of 992 exposure to the study agents will be approximately 4 and one half years (54 months) for a 5 year 993 follow-up interval. 994 5.3 Dose selection – safety and efficacy 995 Both agents have a considerable margin of safety in the doses to be used. The calcium dose of 996 1200 mg has been widely used; in our previous trial, this dose did not confer any toxicity. This 997 dose is very likely to be effective. Our previous clinical trial results, together with human 998 dietary epidemiology, indicate that the calcium dose will be associated with a decreased risk. 999

v 20.0 9-11-14 22

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1000 The vitamin D dose, 1000 IU daily, has been shown to be safe in intervention studies in humans. 1001 Together with the vitamin D in diet (typically 200-300 IU), the total vitamin D intake will be 1002 well below the 2000 IU dose set as the “no observed adverse effect level,” a standard that is 1003 itself conservative. This dose is also likely to be effective. Human epidemiological data suggest 1004 that doses of vitamin D in the range of high dietary intake or common supplemental doses may 1005 decrease the risk of colorectal neoplasia. 1006 1007 5.4 Formulation 1008 We will use calcium carbonate for the calcium supplementation. This compound provides the 1009 convenience of supplying 1200 mg of elemental calcium in two tablets; other calcium salts 1010 would require a larger number of pills. 1011 1012 We will use vitamin D3 for the vitamin D supplementation. This will avoid the greater toxicity 1013 risks associated with 1,25-(OH)2 vitamin D or 25-OH vitamin D. This form of vitamin D is a 1014 pro-hormone that must be metabolized by normal mechanisms in the body to generate the most 1015 active moiety. Supplementation with even large doses of vitamin D3 does not increase total 1016 1,25-(OH)2 vitamin D levels in individuals who are not vitamin D deficient, although toxicity 1017 may still ensue. 1018 1019 Each pill will contain either placebo or elemental calcium (600 mg) and either placebo or 1020 vitamin D (500 IU). 1021 1022 5.5 Administration 1023 The study agents will be taken orally, twice a day, preferably with meals. Food increases the 1024 natural acidity of the stomach thereby enhancing calcium carbonate absorption. 1025 1026 5.6 Side effects 1027 Calcium supplements are generally well tolerated. Use of calcium carbonate reportedly may 1028 cause such gastrointestinal side effects as bloating, gas and flatulence. Prolonged use of large 1029 doses of calcium carbonate –greater than 12 grams daily- may lead to the milk-alkali syndrome, 1030 nephrocalcinosis and renal insufficiency. 1031 1032 Dosage of vitamin D up to 2,400 IU/day in healthy individuals rarely causes adverse reactions. 1033 Chronic use of very high doses (3,800 IU/day or greater) of vitamin D may cause hypercalcemia. 1034 Early symptoms of hypercalcemia include nausea and vomiting, weakness, headache, 1035 somnolence, dry mouth, constipation, metallic taste, muscle and bone pain. Late symptoms and 1036 signs of hypercalcemia include polyuria, polydipsia, anorexia, weight loss, nocturia, 1037 conjunctivitis, pancreatitis, photophobia, rhinorrhea, pruritis, hyperthermia, decreased libido, 1038 elevated BUN, albuminuria, hypercholesterolemia, elevated ALT and AST, ectopic calcification, 1039 nephrocalcinosis, hypertension and cardiac arrhythmias. 1040 1041 5.7 Contraindications 1042 Calcium and vitamin D supplementation are contraindicated in those with hypercalcemia. 1043 Conditions causing hypercalcemia include sarcoidosis, hyperparathryroidism, hypervitaminosis 1044 D and cancer. Calcium and vitamin D supplementation are also contraindicated in those 1045 hypersensitive to any component of the supplement. 1046 1047 5.8 Manufacturer and supplier

v 20.0 9-11-14 23

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1048 Study agents will be manufactured by Pfizer Pharmaceuticals, previously Wyeth Consumer 1049 Healthcare, which will prepare them to our specifications. 1050 1051 5.9 Packaging, labeling and distribution 1052 Run-in and randomized treatment pills will be shipped from Pfizer Pharmaceuticals, previously 1053 Wyeth Consumer Healthcare, to the packaging facility, which will oversee packaging into white 1054 400 cc bottles. The study agent will be packaged to prevent contamination and unacceptable 1055 deterioration during transport and storage. Bottles will be packaged with 250 pills each. The 1056 packaging facility will ship the filled bottles by group and lot number to the Dartmouth Project 1057 Coordination Center’s pharmacy office, where labels will be generated and applied to the bottles 1058 under the supervision of the study pharmacist. 1059 1060 Run-in Pills: Bottles containing run-in pills (placebo or calcium alone) will be labeled with the 1061 study name and bottle number (date). Upon obtaining all required Study Center documentation 1062 (e.g. local IRB approval), the bottled run-in pills will be sent from the Dartmouth Project 1063 Coordination Center’s pharmacy office to the Study Center’s pharmacy or study office for 1064 storage and distribution to participants upon completion of their intake appointment. A space 1065 will be provided on the label for the study coordinator or pharmacist to write the participant’s 1066 name after the bottle has been assigned to a participant. Bottles will be shipped as needed to 1067 ensure an adequate supply at each Study Center for participant enrollment. 1068 1069 First Randomized Pill Bottle: Participants who continue to be eligible for and interested in study 1070 participation at the end of the run-in period will be sent a new supply of study pills from the 1071 Dartmouth Project Coordination Center immediately after the randomization assessment phone 1072 call. The new supply will be the randomly assigned study agent. Subjects will be instructed to 1073 start this new bottle immediately upon receipt and to record the date that the bottle is started. 1074 The randomized pill bottles will be labeled with the participant’s name and shipped directly to 1075 the participant from the Project Coordination Center. 1076 1077 Subsequent Randomized Pills: Subjects will be instructed to finish their old bottle of study pills 1078 prior to starting their new one and to record the date that the new bottle is started. Participants 1079 will be provided a postcard or telephone number to report the date the first pill was taken from 1080 each interval shipment bottle. Bottles containing randomized pills will be mailed in 1081 approximately four month intervals adjusting for the previous pill bottle start date. Special 1082 shipments will also be made upon request, if, for example, a participant loses their bottle or does 1083 not receive their new bottle of pills. 1084 1085 Final Randomized Pills: For subjects still on treatment 5 months prior to the end of the treatment 1086 phase of the study on 12/1/13, the following additional steps will be taken. Participants will 1087 receive a letter with their study pills instructing them to discontinue pill taking as of 12/1/13. 1088 The pill bottle will also be labeled "Discard after 12/1/13". 1089 1090 FDA labeling requirements will also be followed in accordance with Investigational New Drug 1091 regulations, as applicable. 1092 1093 Quality Control. After randomization, pill content will be tested periodically (approximately 1094 annually) under the supervision of the study pharmacist on about 25 random participants in each

v 20.0 9-11-14 24

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1095 of the four treatment groups to monitor errors in agent manufacturing, packaging, labeling and 1096 distribution procedures. 1097 1098 5.10 Storage 1099 Bottles shipped from the packaging facility to the Dartmouth Project Coordination Center will be 1100 stored under the supervision of the study pharmacist in a secure area in the pharmacy office. The 1101 study pharmacist will supervise acceptable storage temperatures, conditions (e.g., protection 1102 from light), and storage times. Bottles shipped from the Dartmouth Project Coordination Center 1103 to the Study Centers (run-in pill bottles only) will be stored under the supervision of the local 1104 pharmacist in a secure area at the center’s pharmacy, or they may be stored under the supervision 1105 of the study coordinator in a secure area at the local study office. The Study Center pharmacist or 1106 coordinator will supervise acceptable storage temperatures, conditions, and storage times. 1107 1108 5.11Blinding and unblinding methods 1109 Neither the staff of the Study Center or the study participants will know the treatment category to 1110 which a participant has been assigned. At the Dartmouth Project Coordination Center, all staff, 1111 except for the study pharmacy staff and some statistical and informatics staff, will also be 1112 blinded to the treatment categories to which participants are assigned. 1113 1114 Upon distribution of the initial randomized pills from the Dartmouth Project Coordination 1115 Center, a sealed envelope for each participant marked “top security”, containing the treatment 1116 assignment, will be sent concurrently to the Study Center under the supervision of the 1117 pharmacist or study coordinator. 1118 1119 It may be necessary to break the blinding for a study participant if he/she develops 1120 hypercalcemia or symptoms of hypercalcemia or if his/her physician requests this information, 1121 generally in an emergency situation. In this event, designated Study Center personnel may open 1122 a participant’s envelope to determine his/her treatment group. Procedures will be provided to the 1123 staff for opening, reporting and returning opened envelopes to the Dartmouth Project 1124 Coordination Center. At the end of the study, the unopened envelopes will be returned to the 1125 Project Coordination Center for inspection and destruction. 1126 1127 Subjects may be notified of their randomized treatment assignments upon completion of all 1128 follow-up colonoscopies at Year 5. At that time all study staff may also be unblinded to 1129 treatment assignments. 1130 1131 5.12 Adherence/compliance 1132 Adherence to treatment will be assessed in three ways: 1) Participant-reported compliance: every 1133 6 months, during the telephone interview with the study coordinator, the participant will be 1134 asked to report how many pills they took per week on average since their last phone interview. 1135 A report of 14 pills would represent 100 % compliance, 13 pills would represent 93 % 1136 compliance, etc... 2) Computed compliance based on participant-reported pill bottle start dates: 1137 participants will be asked to record and report to their study coordinator the date that they start 1138 each new bottle of study pills. Given the start date of each pill bottle and the total number of 1139 pills in each bottle (which the participant will be instructed to finish prior to starting the next 1140 bottle) we will calculate a compliance rate for each bottle. 3) Serum 25-OH vitamin D levels 1141 will be measured at baseline and at Years 1 and 3, and, where applicable, at year 5. To maintain 1142 blinding, these data will only be used to measure compliance at the level of treatment groups.

v 20.0 9-11-14 25

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1143 The Project Coordination Center will regularly summarize the pill-taking status of each center's 1144 subjects, and list for each coordinator the relevant study ID numbers of those who have reported 1145 changed or inadequate adherence. 1146 1147 5.13 Agent accountability 1148 The Dartmouth Project Coordination Center and the Study Centers, through written standard 1149 operating procedures, will ensure the adequate and safe receipt, handling, storage, dispensing, 1150 and return and/or disposal of unused study agents by study staff and study participants in 1151 compliance with applicable regulatory requirements. 1152 1153 5.14 Agent disposal 1154 The Dartmouth Project Coordination Center and the Study Centers, through written standard 1155 operating procedures, will maintain a system for the disposal of unused or expired study agents 1156 and for the documentation of this disposal. 1157 1158 5.15 Assessment of blinding effectiveness 1159 In a randomized placebo-controlled trial, the validity of the results depends on the effectiveness 1160 of methods to blind participants to the randomization assignment. Unblinding is a potential 1161 source of bias because someone who believes they are getting the active treatment may 1162 experience or report better outcomes. To assess unblinding, this study includes a longitudinal 1163 evaluation of blinding effectiveness as well as an assessment of the effect of participants' beliefs 1164 about the intervention on measured and self-reported health outcomes in each intervention arm. 1165 Participants will be asked at Intake which pill they would prefer to receive if they had a 1166 choice. They will also be asked to describe their beliefs about the efficacy of calcium and 1167 vitamin D in relation to general health, prevention of colorectal polyps, and improvement of 1168 mood, depression, constipation, and bone and joint pain. After the run-in period they will be 1169 asked to guess which pill they think they have received and the reasons for their guess. In 1170 addition, subjects will be asked to complete two more questionnaires, by mail or telephone 1171 interview, at the end of year 2 and at the time of the participants' follow-up colonoscopy (Year 3 1172 or 5). These questionnaires will be administered by independent staff from Geisel School of 1173 Medicine at Dartmouth and not by the study coordinators, because the coordinators help to 1174 collect endpoint data and could be biased by participants' responses. 1175 1176 6.0 Post-randomization Follow-up 1177 1178 The study coordinators at each Study Center will maintain regular contact with all subjects. 1179 Scheduled contacts with the subject are listed below. Some subjects, however, will be contacted 1180 more frequently to encourage adherence to treatment or to manage individual problems. 1181 1182 Follow-up activities once a subject is randomized will include: 1183 • Every six months - interval questionnaire 1184 • At years 1 and (where applicable) year 3 - laboratory measurements of calcium, 1185 creatinine, and vitamin D 1186 • At Year 3 or Year 5 – follow-up colonoscopy, SF-36 Health Survey, End of 1187 Treatment Questionnaire, and laboratory measurement of vitamin D 1188

v 20.0 9-11-14 26

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1189 All participants who complete Year 3 or Year 5 follow-up activities will be reimbursed $100 to 1190 defray the time and expense of these activities. 1191 1192 6.1 Interval Questionnaires 1193 Every 6 months, all subjects will have a telephone interview regarding somatic symptoms 1194 possibly related to study agents, illnesses, doctor’s visits, hospitalizations, medications, over-the- 1195 counter preparations (including nutritional supplements), dietary intake of calcium and vitamin 1196 D, and compliance with study tablets. The responses to these questions will be entered by the 1197 study coordinator into a secure web-based form that transmits the data directly to the Dartmouth 1198 Project Coordination Center in real-time over the Internet. If access to the internet is interrupted 1199 or temporarily unavailable, a paper form will be used and the data will subsequently be entered 1200 into the web form. The interval questionnaire interview may also be performed in person when 1201 in conjunction with a Year 1 or Year 3 blood draw, as appropriate. If necessary (e.g., a subject is 1202 unavailable by phone or deaf), a subject may self-complete a paper questionnaire and return it to 1203 the study coordinator for data entry into the web form. Finally, subjects who have access to the 1204 internet will be able to self-complete the questionnaire on the web once that technology is 1205 available in the study. The interval questionnaire should be collected within 30 days on either 1206 side of each 6 month anniversary of the randomization date. 1207 1208 6.2 Schedule of Lab Measurement 1209 Laboratory measurements at years 1, 3 and 5 will be used for safety and compliance purposes. 1210 The schedule of laboratory tests are summarized in the following table for subjects with either 3 1211 or 5 year follow-up colonoscopies. 1212 Lab Measurement Entry Year 1 Year 3 Year 5 Calcium XO XO O - Creatinine XO XO O - 25-(OH)-vitamin D XO XO XO O 1213 1214 Key: Subjects scheduled for colonoscopy follow-up exam at 3 years: X 1215 Subjects scheduled for colonoscopy follow-up exam at 5 years: O 1216 1217 The interim blood draw(s) will be performed 1 year after randomization, as well as 3 years after 1218 randomization for subjects with a 5-year surveillance cycle. The interim blood draw(s) will be 1219 collected within 30 days prior to and 90 days after the randomization anniversary date. If the 1220 total calcium measurement is slightly elevated or slightly low, a re-test (of the original blood 1221 draw specimen) or a re-draw (draw for a new blood specimen), including an albumin 1222 measurement, will be used to estimate the ionized calcium level. 1223 1224 The final blood draw (year 3 or year 5) will be within the 60 days prior to the follow-up 1225 colonoscopy, while the subject is still taking the study pills, and, preferably, should not be 1226 performed at the colonoscopy exam (e.g. after the colonoscopy preparation has been 1227 administered to the subject). 1228 1229 As already covered in the informed consent form, additional blood draw(s) will be performed in 1230 the event re-testing is required in follow up to lab results or in circumstances of overdue 1231 colonoscopy when the subject wishes to continue study treatment.

v 20.0 9-11-14 27

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1232 1233 If the 25-(OH)-vitamin D value is out-of-range (less than 10 or more than 100 ng/ml) then a 1234 sample from the same blood draw will be re-tested at a clinical laboratory that is certified per 1235 CLIA (Clinical Laboratory Improvement Amendments). If the re-test value is less than 10 or 1236 more than 100 ng/ml then the subject and their health care provider will be notified of the result. 1237 1238 6.2.1 In follow up to an Unanticipated Problem Report of October 10, 2011: 1239 1240 Additional study blood draws and tests will be completed so that results can be utilized in 1241 ongoing data analyses and subject notification via participant materials for this purpose. 1242 1243 1. Subjects still on study treatment will have annual blood draws for creatinine 1244 measurement. Those whose levels are greater than the upper limit of normal will not 1245 continue study calcium treatment. Calcium levels will also be measured at these blood 1246 draws, as funding permits. As a result of these new procedures, the window for the “End 1247 of Treatment visit” and blood draw will be expanded to within approximately one year of 1248 the anticipated follow-up colonoscopy exam. Subjects will also be asked a new question 1249 regarding their last use of study pills or calcium supplements at this visit. 1250 1251 2. Subjects who have already completed the treatment phase of the study will have 1252 creatinine measured on stored blood specimens drawn at the “End of Treatment Visit.” 1253 Calcium, and other analytes related to kidney function, will also be measured on these 1254 stored samples, as funding permits. 1255 1256 3. Subjects who have completed the treatment phase of the study, and whose last study 1257 creatinine levels are above the normal lab range will be invited to return for a blood draw 1258 to re-measure the levels. Calcium levels will also be measured at these blood draws, as 1259 funding permits. In addition, these subjects will be asked to complete a health update 1260 using the current Observational Follow Up Questionnaire and will also be asked a new 1261 question regarding their last use of calcium supplements. A separate new Informed 1262 Consent form will be used to obtain subject consent for this additional study visit (the 1263 “Post-End of Treatment Additional Study Visit Consent”). 1264 1265 4. Subjects will be sent materials describing this new development. All subjects who were 1266 randomized into the study will be sent these materials with the sole exception of deceased 1267 subjects. The materials include: 1268 a. Letter to Subject with Normal Creatinine Result 1269 b. Letter to Subject with Above Normal Creatinine Result 1270 c. Above normal creatinine result with specific result and lab range 1271 d. Significant New Information/Findings sheet for Pre-EOT subjects 1272 e. Significant New Information/Findings sheet for Post-EOT subjects 1273 f. Post-EOT Additional Study Visit Consent 1274

v 20.0 9-11-14 28

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1275 5. Subjects who choose to discontinue study calcium treatment will be urged to continue 1276 study vitamin D treatment. 1277 1278 6.2.2 April 11, 2012 modification to the creatinine criteria and procedures to be used due to the 1279 Unanticipated Problem 1280 1281 1. Because many of the local laboratories used for creatinine measurements in the study do 1282 not take sex, age or race into account, the creatinine reference ranges provided by those 1283 laboratories may not be appropriate for the individual subject. Therefore, the criteria for 1284 stopping study calcium treatment (randomized or two-arm) has been changed to an 1285 increase of 0.3 mg/dL or more from baseline (update to #1 in section 6.3.1 above, 1286 originally implemented in October 2011). 1287 1288 2. Similarly, subjects who complete or who already have completed the treatment phase of 1289 the study and whose last creatinine level in the study was increased by 0.3 mg/dL or 1290 more from baseline will be invited to return for a blood draw to re-measure the levels 1291 (update to #3 in section 6.3.1 above, originally implemented in October 2011). 1292 1293 3. In place of annual study blood draws during the treatment phase or a study visit after 1294 completion of the treatment phase, creatinine (and calcium) levels may be documented 1295 from medical records. 1296 1297 6.3 Colonoscopy 1298 Colonoscopic visualization of the entire large bowel will be performed in each subject 1299 approximately 36 months or 60 months after the qualifying colonoscopy, as set out before 1300 randomization by each patient's clinician. This time frame (3 or 5 years) for colonoscopy 1301 surveillance following the qualifying examination is consistent with current clinical guidelines. 1302 Additional exams will not be encouraged unless required clinically; patients in whom more 1303 frequent exams are anticipated will not be randomized. Study coordinators will contact subjects 1304 by telephone or mail to remind them of their anticipated examination and to assist in scheduling, 1305 as possible. Study coordinators will contact subjects who do not return for examinations and 1306 attempt to persuade them to have their follow-up colonoscopy or determine why this is not 1307 possible. The specific timing of this follow up colonoscopy, which results in the end of the 1308 treatment phase of the study for this subject, will vary according to the circumstances of the 1309 individual subject and the scheduling procedures of the clinic and the patient’s clinician. 1310 Therefore, the window for this colonoscopy will be from 6 months prior to the 3 or 5 year 1311 anniversary of the qualifying colonoscopy exam and up to the End of Treatment Phase as 1312 specified in 6.8 below . Colonoscopies performed prior to this window may be considered 1313 interim exams, and study treatment may continue. 1314 1315 6.4 RAND SF-36v2 Health Survey 1316 In addition to at enrollment, subjects will also be asked to complete an SF-36v2 health survey 1317 two additional times during the study--at the end of year 2 and at the time of the three-year or 1318 five-year follow-up colonoscopy exam. This 36-item self-administered questionnaire was 1319 constructed to survey health status in the Medical Outcomes Study. It was designed for use in 1320 clinical practice and research and has been extensively tested and validated. It assesses eight 1321 different health concepts: 1) limitations in physical activities because of health problems; 2)

v 20.0 9-11-14 29

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1322 limitations in social activities because of physical or emotional problems; 3) limitations in usual 1323 role activities because of physical health problems; 4) limitations in usual role activities because 1324 of emotional problems; 5) general mental health (psychological distress and well-being); 6) 1325 bodily pain; 7) vitality (energy and fatigue); and 8) general health perceptions. 1326 1327 6.5 End of Treatment Visit 1328 An End of Treatment Visit will be conducted within the 60 days prior to the follow-up 1329 colonoscopy. Study coordinators will contact subjects by phone or mail to schedule this visit. 1330 Prior to the scheduled visit, a packet (#4) will be sent to the subject with the following items: 1331 • Letter confirming the appointment date and time 1332 • Checklist for the appointment 1333 • RAND SF-36 Health Survey 1334 • Copy of the Observational Follow Up Informed Consent document 1335 1336 The End of Treatment Visit will include a final blood draw (see section 6.3 above), collection of 1337 the SF-36 Health Survey, and measurement of height and weight. In addition, the Observational 1338 Follow Up phase of the study will be described (see section 6.7 below). If the subject is 1339 interested in participating in the Observational Follow Up, then the informed consent process 1340 will be carried out and consent will be documented by the subject’s signature on the informed 1341 consent form. 1342 1343 6.6 End of Treatment Questionnaire 1344 At the end of treatment, all subjects will complete a final questionnaire that captures the same 1345 information collected on the Interval Questionnaire (symptoms, illnesses, doctor’s visits, 1346 hospitalizations, medications, over-the-counter preparations, dietary intake of calcium and 1347 vitamin D, and compliance with study tablets) as well as some additional information (exercise, 1348 smoking, bowel movements, and postmenopausal status). In addition, the name of a contact 1349 person will be collected to assist, if necessary, in locating a subject who can not be found at a 1350 later date. The End of Treatment Questionnaire will be collected within 60 days of the follow up 1351 colonoscopy that results in the end of treatment for that subject. 1352 1353 6.7 End of the Treatment Phase 1354 The Treatment Phase of the study will end on August 31, 2013, after which subjects who have 1355 not already completed the Treatment Phase will be instructed to discontinue pill taking. 1356 1357 After the Treatment Phase ends, a packet will be mailed to subjects, including the following 1358 items: 1359 1360 • Treatment Assignment Notification from the Project Coordination Center at Dartmouth 1361 • Participant Survey with self-addressed and stamped envelope for returning the completed 1362 survey to the Project Coordination Center at Dartmouth 1363 1364 When the main study Treatment Phase findings are first presented at a national meeting or 1365 published, all subjects will be notified of the findings by letter. 1366 1367 6.8 Observational Follow Up

v 20.0 9-11-14 30

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1368 Subjects will be invited to participate in an optional Observational Follow Up phase of the study 1369 that will begin following the end of treatment. In this phase of the study, subjects will continue 1370 to be followed on an observational basis (no study treatment) with annual questionnaires until 1371 the time of a subsequent colonoscopy that is at least three years from the follow up colonoscopy 1372 at which study treatment was ended. 1373 Annual questionnaires will be used to collect updated information on the subject’s 1374 health, medical care, use of medications or supplements, and diet. These questionnaires will be 1375 collected within 30 days before or after the annual anniversary date of the end of treatment 1376 colonoscopy. The questionnaires may be collected by coordinator interview of the subject 1377 (preferred), or by subject self-completion of a paper form or a web-based form (once that 1378 technology is available in the study). Medical records will be obtained to confirm any medical 1379 events reported by the subject. 1380 For any colonoscopies that are reported by the subject, medical records will be obtained 1381 and any pathology specimens will be obtained and reviewed by the Study Pathologist as 1382 described below (section 8.0) for pathology collected during the treatment phase of the study. 1383 Observational follow up will continue until a colonoscopy is performed that is at least 3 years 1384 from the end of treatment for that subject. Within 60 days of that colonoscopy, a final 1385 questionnaire will be collected and the subject’s participation in the Observational Follow Up 1386 phase of the study will end. However, if a colonoscopy is not performed byJuly 31, 2016, which 1387 is the end of the study, then observational follow up will end at that time. 1388 1389 7.0 Clinical Procedures – Endoscopy 1390 1391 The endpoint of the study will be new adenomas detected on follow-up colonoscopy. 1392 Colonoscopic visualization of the entire large bowel will be performed in each subject 1393 approximately 3 or 5 years after the qualifying colonoscopy, as set out before randomization by 1394 each patient's clinician. 1395 1396 7.1 Overview of Procedure 1397 The coordinator at each Study Center will provide study colonoscopy guidelines to the 1398 endoscopist prior to each study exam. Participants will undergo pre-colonoscopy bowel 1399 preparation using a standard oral lavage or phospha-soda bowel cleansing preparation, according 1400 to the preference of the local study colonoscopist. On the day of the examination each subject 1401 will be asked to sign a routine informed consent document for the procedure as required by each 1402 institution. Prior to colonoscopy, each subject may receive intravenous medication to achieve 1403 desired conscious sedation following the usual practice of the endoscopist performing the 1404 procedure. Continuous monitoring of the patient will be carried out by the endoscopy unit 1405 nursing personnel before, during, and after the procedure until adequate recovery, according to 1406 the standards and practice protocols of the endoscopy unit. 1407 1408 Colonoscopy to the cecum will be performed using standard techniques. The entire colonic 1409 mucosa will be carefully examined during withdrawal of the instrument. Detected polypoid 1410 lesions will be sized in comparison to open biopsy forceps and then resected using standard 1411 biopsy forceps or snare techniques, avoiding cautery if possible. The location of each lesion will 1412 be recorded by segment of the colon and centimeters from the anus, and will be submitted to the 1413 local pathology laboratory in a separate specimen bottle. Colonoscopic findings will be recorded 1414 on the colonoscopy/surgery reporting form immediately after the procedure. If at colonoscopy

v 20.0 9-11-14 31

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1415 the preparation is judged to be inadequate, or if the colonoscopy is incomplete for other reasons, 1416 a second colonoscopy should immediately be scheduled. 1417 1418 The endoscopist will inform each subject of findings at the time of the procedure. If polyps or 1419 masses are found, the participant will be contacted by an appropriate clinician with the pathology 1420 findings as soon as they are available, and any required follow-up treatment will be arranged. 1421 Any other follow-up required by a participant, including care of complications, will be handled 1422 at each Study Center by the endoscopist and/or other medical staff according to the usual 1423 practice of the endoscopy unit. 1424 1425 7.2 Quality Assurance 1426 Endoscopy guidelines in the form of laminated pocket-sized cards for physicians will be 1427 provided by the Dartmouth Project Coordination Center. The card will be personalized for each 1428 center, giving the study coordinator’s phone number so that physicians can call them with any 1429 questions or problems. Current Polyp Prevention Study guidelines for endoscopists include the 1430 following: 1431 • Notation of the adequacy of the bowel prep 1432 • Verification of the completeness of colonoscopy by documentation of at least 2 usual 1433 landmarks, e.g., cecal strap, appendiceal orifice, ileocecal valve. 1434 • Biopsy of all mucosal excrescences and lesions suspicious for neoplasia. Avoidance of 1435 fulguration without biopsy. 1436 • If clusters of diminutive (<5mm) polyps are noted in the distal rectum, removal of all of 1437 the polyps or a representative sample (to a maximum of 5) and documentation as 1438 accurately as possible of the number of any remaining polyps (a range of estimated 1439 numbers if necessary). 1440 • Placement of each lesion specimen in a separate pathology bottle. 1441 1442 8.0 Endpoint Determinations and Pathology 1443 1444 Colorectal adenomas are the study’s main endpoint, so they will be ascertained completely and 1445 accurately. At all endoscopic examinations after randomization, all raised mucosal lesions, and 1446 all flat lesions suspicious for neoplasia, will be biopsied and excised, their location and size 1447 noted, and pathology material reviewed by the study pathology center for confirmation of 1448 diagnosis. If clinical events lead to interim endoscopy, all the relevant information and 1449 histological material will be obtained from that endoscopy as well. For each subject, the number 1450 of mucosal lesions will be tabulated, along with their size, histological characteristics, and 1451 location in the bowel. 1452 1453 Tissue biopsies of colon and rectal polyps will be collected using standard procedures during 1454 endoscopies performed by qualified endoscopist at each of the Study Centers. If at all possible, 1455 each polyp specimen will be put into a separate pathology bottle for processing. Slides will be 1456 prepared at the local Study Center pathology labs according to standard procedures. Study 1457 coordinators will purchase or request loans of these slides from the pathology labs for analysis 1458 by our study pathologist. These slides will be shipped to the Dartmouth Project Coordination 1459 Center on a quarterly basis. Slides will be stored at room temperature in a secure (locked) office 1460 at Dartmouth prior to batch shipments to the study pathologist (Dr. Dale Snover at Fairview 1461 Southdale Hospital in Edina, Minnesota). After review by the study pathologist, the slides will

v 20.0 9-11-14 32

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1462 be returned to the Dartmouth Project Coordination Center and, if on loan, then returned to the 1463 Study Center of origin, unless the subject consents to their storage for future research and 1464 permission is obtained from the pathology laboratory of origin. All pathology slides that are 1465 collected during this polyp prevention trial will be tracked electronically from the time of 1466 shipment to the Project Coordination Center to the time of return to the Study Center of origin. 1467 The tracking number used will be the local pathology ID number that is found on the slide. An 1468 archive of slides purchased by the study will be maintained at the Project Coordination Center as 1469 part of the study records. 1470 1471 Digital microscopy is increasingly being used for histology specimens. Thus, whenever 1472 possible, digital images will be made available to the Study Pathologist instead of the slides 1473 themselves. Depending on the availability of the requisite scanning equipment, the digital 1474 images may originate at the Study Centers, or may be created at the Dartmouth Project 1475 Coordination Center (after having obtained the slides from the Centers without this equipment). 1476 The images will be made available to the Study Pathologist via the internet, or via mailed 1477 DVD's. An archive of the images will be maintained at the Project Coordinating Center as part 1478 of the study records. 1479 1480 The study pathologist will review all pathology specimens (slides or digital images) without 1481 knowledge of treatment assignment. Each polyp will be classified (using standard criteria) as 1482 neoplastic (tubular, tubulovillous, villous, or serrated adenomas or cancer), hyperplastic, 1483 hamartomatous, retention, lymphoid, or other. In addition, for the neoplastic polyps, a statement 1484 with regard to the degree of atypia present will be made using standard criteria. When there is 1485 disagreement between the local and study readings, we will first ascertain that the study 1486 pathologist has read the particular specimen (slide or digital image) used to make the local 1487 diagnosis and will send further specimens if appropriate. If disagreement remains, we will 1488 accept the study pathologist's diagnosis. 1489 1490 9.0 Laboratory Evaluations – Blood Samples 1491 1492 9.1 Laboratories 1493 Creatinine, total calcium, and albumin measurements will be performed at local clinical 1494 laboratories associated with each of the participating Study Centers or their recruitment sites. 1495 1496 Serum vitamin D measurement will be conducted at the University of California - Los Angeles 1497 (UCLA) Nutrition Research Center Laboratory by Dr. David Heber. This is a research 1498 laboratory that is not certified per CLIA (Clinical Lab Improvement Amendments). Therefore, 1499 the results of these measurements may not be used in clinical decision making and will not be 1500 disclosed to subjects or to their health care provider. However, if the 25-(OH) vitamin D value 1501 at an interim or final study blood draws is out of range (below 10 or more than 100 ng/ml) then 1502 the blood sample will be re-tested at a CLIA certified laboratory. Mayo Medical Laboratories 1503 (Rochester, MN) is being used for this purpose. 1504 1505 DNA extraction from buffy coat samples and VDR genotyping will be conducted in Dr. Robert 1506 Haile’s laboratory at the University of Southern California in Los Angeles or in another qualified 1507 collaborating or commercial laboratory. 1508

v 20.0 9-11-14 33

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1509 Blood samples that will be used for DNA isolation and for future research will be stored at the 1510 Norris Cotton Cancer Center’s (NCCC) Clinical & Molecular Pharmacology Shared Resource 1511 Laboratory in Lebanon, New Hampshire. 1512 1513 9.2 Collection and Handling Procedures 1514 Whenever possible, subjects should be fasting prior to all study blood draws (e.g. no food or 1515 beverage intake other than water for 8 hours prior to the blood draw). Regardless, the time of 1516 last food or beverage consumption will be collected. Customized prepackaged bar-coded kits 1517 will be provided containing materials for collecting, processing, and aliquoting the blood 1518 samples. Blood samples will be collected according to standard operating procedures at each 1519 Study Center and sent to local labs for analysis or promptly frozen at -20°C or below pending 1520 transfer to a -70°C freezer for storage until shipment to the UCLA Nutrition Research Center 1521 Laboratory or the NCCC Clinical & Molecular Pharmacology Shared Resource Laboratory. 1522 Each individual blood specimen will be bar-coded and tracked electronically from the time of 1523 collection at the Study Center. Biospecimen transport will adhere to stringent shipping 1524 standards. The Study Centers, study laboratories (at UCLA and USC), and the biospecimen 1525 storage facility at the NCCC will be equipped with at least one bar code reader for logging and 1526 tracking specimens. 1527 1528 As necessary for all study blood draws, a re-draw may be required to collect blood if the original 1529 specimen is lost, destroyed, damaged, or if a re-analyses is required (e.g., albumin measurement 1530 to estimate ionized calcium at Year 1 or Year 3 safety assessment). 1531 1532 9.3 Methods for Laboratory Procedures 1533 1534 Measurement of Calcium and Creatinine 1535 Blood levels of creatinine, total calcium, and albumin will be measured according to standard 1536 procedures at the local clinical laboratories. The normal reference ranges of the local 1537 laboratories will be used for eligibility assessment at enrollment and safety assessment at year 1 1538 and, where applicable, year 3 (for those with a 5 year follow-up colonoscopy interval). 1539 1540 Measurement of Vitamin D 1541 25-Hydroxy-Vitamin D Determination: 1542 The Gamma-B 25-hydroxy-vitamin D RIA kit from IDS Inc. (Fountain Hills, AZ) is used to 1543 determine serum concentrations of 25-hydroxy-vitamin D. The IDS gamma-B 25-OH-vitamin D 1544 kit is a liquid phase radioimmunoassay. The addition of reagent 1 and 2 causes the precipitation 1545 of serum proteins and 25-hydroxy-vitamin D remains in solution. Following centrifugation, 1546 portions of the supernatant are incubated with 125I labeled 25-hydroxy-vitamin D and a highly- 1547 specific sheep antibody to 25-hydroxy-vitamin D. Separation of antibody-bound tracer from free 1548 is achieved by a short incubation with Sac-Cel (anti-sheep IgG cellulose) followed by 1549 centrifugation and decanting. Bound radioactivity in the Sac-Cel pellet is inversely proportional 1550 to the concentration of 25-hydroxy-vitamin D. The percent binding is determined for several 1551 standard concentrations and a calibration curve is established. Percent binding of the unknown 1552 samples is calculated and concentration determined from the calibration curve. For quality 1553 control purposes, two kit controls are analyzed with every batch. In addition five unknown 1554 samples from the international Vitamin D External Quality Assessment Scheme (DEQAS)

v 20.0 9-11-14 34

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1555 program are analyzed with every kit. The intra assay correlation coefficient is 5.3%-6.1% (26.5- 1556 151 nmol/L) and inter assay correlation coefficient is 8.2-7.3% (19.6-136 nmol/L). 1557 1558 Measurement of VDR polymorphisms 1559 Very briefly, DNA will be extracted and VDR genotyping will be conducted by PCR analysis 1560 using gene-specific primers. 1561 1562 1563 10.0 Safety Monitoring and Reporting Adverse Events 1564 1565 10.1 Definition, Monitoring and Follow-up 1566 An adverse event is any condition that appears or worsens after the subject is enrolled in an 1567 investigational study. Adverse events may be discovered as a result of study lab measurements 1568 or during regular study interviews (every six months). As a safety measure, blood levels of 1569 calcium, creatinine and 25-OH vitamin D will be monitored one year, and where applicable three 1570 years after randomization. At each regularly scheduled interval contact (every six months) study 1571 coordinators will monitor symptoms, medical diagnoses and hospitalization as part of the subject 1572 case report form. Adverse events may also be discovered by subject-initiated contact at any 1573 time. All adverse events that meet study specific criteria are documented on an Adverse Event 1574 Report Form. There are three types of events that have been defined: Perceived Toxicity, 1575 Diagnosis and Out-Of-Range Lab Result. All adverse events, including laboratory abnormalities 1576 that in the opinion of the Clinical Director are clinically significant, will be followed up 1577 according to good medical practice, and documented as such. 1578 1579 10.2 Adverse Event Information and Severity 1580 The following information will be collected on the Adverse Event Report Form: start and stop 1581 dates, severity (grade), relationship to study agents, and whether or not the subject stopped 1582 taking the study agents due to the adverse event. Severity will be graded by a numerical score 1583 according to the NCI Common Terminology Criteria for Adverse Events (CTCAE - version 3.0 1584 or newer). Events not included in the defined NCI CTCAE will be scored according to their 1585 impact on the subject’s abilities to perform daily activities as follows: 1586 a. Mild (causing no limitation of usual activities) 1587 b. Moderate (causing some limitation of usual activities) 1588 c. Severe (causing inability to carry out usual activities) 1589 1590 10.3 Serious Adverse Events 1591 Serious adverse events are defined as those events that meet any of the following criteria: 1592 • Results in death 1593 • Is immediately life threatening 1594 • Requires inpatient hospitalization or prolongation of existing hospitalization 1595 • Results in persistent or significant disability or incapacity 1596 • Is a congenital anomaly or birth defect 1597 1598 10.4 Reporting 1599 Adverse events will be reported to study center IRBs according to local IRB policy. Study 1600 Centers are responsible for reporting all adverse events to the Project Coordination Center 1601 according to the requirements and timelines set forth in the standard operating procedures for

v 20.0 9-11-14 35

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1602 this study. Unexpected serious adverse events possibly related or related to the study agents will 1603 be reported on an expedited basis to the NIH, the FDA (as required by law), Pfizer 1604 Pharmaceuticals, previously Wyeth Consumer Healthcare, and the Safety and Data Monitoring 1605 Committee. In addition, every six months the Safety and Data Monitoring Committee will 1606 monitor study-wide adverse events and the overall conduct of the trial. Subsequently, a report 1607 summarizing study-wide adverse events will be sent to each Study Center for submission to the 1608 individual IRBs. 1609 1610 1611 11.0 Data Management 1612 1613 11.1 Central database 1614 The central database was developed using a SQL-based database management application and 1615 will be administered by the Informatics group within the Section of Biostatistics and 1616 Epidemiology at Geisel School of Medicine at Dartmouth. Any data errors or inconsistencies 1617 detected after data entry will be automatically tracked, communicated and resolved using the 1618 Informatics center's web-based application. An audit trail of all data changes over the life of the 1619 study will be maintained. The database is located on a server protected by firewalls using 1620 stateful inspection firewalling and protocol and application inspection. Access to the database 1621 server will no be allowed by users on computers outside of the firewall protected zone. 1622 1623 11.2 Website 1624 This study will employ real-time web-based data entry at the Study Centers. A central study 1625 website will provide ready and secure access to entry interfaces and current study information 1626 including contact information for all study personnel, the study protocol, policies and 1627 procedures, updated forms and questionnaire versions, and real-time reports. The web server 1628 will run Apache on Linux OS and Microsoft IIS Server on Windows NT, providing a high level 1629 of security through authentication and 128 bit encryption using SSL. The website was 1630 developed using HTML, Java, Javascript, and PDF-formatted documents, and will be accessible 1631 from all current browsers. A separate study web page will be used to provide access to subjects 1632 for self-completion of interval questionnaires. 1633 1634 11.3 Data Entry 1635 Most data entry will occur when the study coordinator is able to interview subjects in a location 1636 with a networked computer so that data can be entered in real time into forms or questionnaires 1637 served from the Dartmouth web server. The study coordinator will access the database interface 1638 through the study URL, be authenticated, link to the appropriate form, and enter data directly 1639 into the central database. Data will be checked in real time via scripted validation for range, data 1640 type, field length, and logical consistency with related data fields. These routines will prompt 1641 the coordinator to reconcile inappropriate or inconsistent data, and will not permit missing 1642 mandatory data. Critical data (such as study identification number) will be double-keyed. 1643 Additionally, to reduce entry error, appropriate data will be pulled from related tables within the 1644 central database and will be used to create pull-down menus and pick-lists for data entry 1645 whenever possible. Study coordinators will be encouraged to use this electronic (validated) data 1646 entry whenever possible. Subjects will also be able to enter data directly into interval 1647 questionnaires on the web. 1648

v 20.0 9-11-14 36

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1649 If internet access is temporarily disabled or unavailable, a low-tech backup (paper forms) will be 1650 provided to ensure timely data collection under any circumstance. All data that is collected on 1651 paper forms will subsequently be entered into the electronic study forms on the web by the study 1652 coordinator. Electronic data entry must occur within 7 days of data collection. Double entry 1653 will be required. 1654 1655 Electronic study questionnaires, which are administered to subjects by their study coordinator, 1656 will include the intake questionnaire (to be used at the intake appointment), the first phone 1657 interview questionnaire (to be used during the initial phone interview at randomization 1658 assessment), the interval questionnaire (to be used during interviews mainly by phone), every six 1659 months throughout the study, the end of treatment questionnaire, and the observational follow up 1660 questionnaire. Paper versions of these questionnaires are provided for completion by subjects as 1661 necessary (post randomization only), and for IRB review. 1662 1663 11.4 Computer Security and Safety 1664 All computer systems and programs will be password protected, and all electronic 1665 communications of study and other confidential information will be encrypted. Good computer 1666 security practice (restricting physical access to machines, prohibition of password sharing, 1667 logging off computers after work hours or when away from the machine) will be required of all 1668 study personnel. Virus protection software will be installed on each study machine. The virus 1669 detection tools will be used, maintained, audited and, if necessary, updated on all computers and 1670 pathways into the system. All computer systems will be updated regularly with any applicable 1671 operating system and application security patches. System access to computer systems will be 1672 audited. Redundant backups and off-site backup storage will allow for quick restoration of data 1673 in the unlikely event that a hardware failure, disaster, or security breach should occur. Servers 1674 and backups will be located in a secured location with access limited to authorized personnel. 1675 1676 11.5 Archiving 1677 All study raw data, forms, documents, software programs, software applications and computer 1678 data files will be indexed and archived routinely. Strict version control of documents and 1679 software applications will be instituted. Retention of study documentation after study 1680 completion will conform to FDA and NIH requirements. 1681 1682 11.6 Reports 1683 Standardized study management reports will be generated monthly during the recruitment phase 1684 of the study and thereafter at least every quarter for the Project Coordinator at the Dartmouth 1685 Project Coordination Center. These reports will be used to track study progress including 1686 subject enrollment, randomization, compliance, subject status changes, and study events. The 1687 data will be reported for each Study Center individually and summarized for the study as a 1688 whole. 1689 1690 Every six months, a standardized report will also be generated for the Safety and Data 1691 Monitoring Committee meeting. This report will include additional information on clinical 1692 events and adverse events that is coded by blinded treatment group. Other than the study 1693 statistician and statistical analyst, no study personnel will see this report. 1694 1695 1696 12.0 Statistical Methods

v 20.0 9-11-14 37

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1697 1698 12.1 Hypotheses 1699 Our main hypotheses are: 1700 1. Supplementation with vitamin D3 (1000 IU/day) reduces the risk of new adenomas in patients 1701 with a recent history of these tumors. 1702 2. Supplementation with calcium carbonate (1200 mg elemental calcium/day) reduces the risk of 1703 new adenomas 1704 3. Supplementation with both vitamin D3 (1000 IU/day) and calcium carbonate (1200 mg 1705 elemental calcium/day) reduces the risk of new adenomas more than supplementation with 1706 calcium carbonate alone. 1707 1708 Our secondary hypotheses are: 1709 4. Supplementation with vitamin D3, or with vitamin D3 or with calcium carbonate, reduces the 1710 risk of new advanced colorectal lesions (colorectal cancer and adenomas with an estimated 1711 diameter of one centimeter or greater, those with tubulovillous or villous histology, or those 1712 with advanced dysplasia). 1713 5. Supplementation with vitamin D3 will have a greater effect among individuals whose initial 1714 serum 25-OH vitamin D level is less than the overall study median, compared with subjects 1715 whose levels are greater than the median. 1716 6. The effect of supplementation with vitamin D3 will be modified by polymorphisms at the 3' 1717 end of the vitamin D receptor gene. 1718 1719 12.2 Sample Size Justification 1720 We originally anticipated enrolling about 2500 participants to reach a total of approximately 1721 2000 randomized subjects (original power calculations are shown in Table 1 - part A). 1722 Subsequently these numbers were increased to up to 3000 enrolled and up to 2400 randomized in 1723 order to increase representation of women, minorities (including African Americans and 1724 Hispanics) and high-risk subjects (defined as subjects with advanced adenomas or multiple (>2) 1725 adenomas at baseline). These updated power calculation are shown in Table 1 (part B) and 1726 described below. Based on our previous trials, we expect that at least 80% of subjects will 1727 complete the run-in period, leaving 2400 patients randomized. Of these, we expect that 60% will 1728 be male and 40% female. Assuming that 35% of females agree to be randomized to calcium, we 1729 expect to have 1776 subjects randomized in the full factorial design and an additional 624 1730 women (taking calcium) randomized to vitamin D or placebo. In our previous trials, we obtained 1731 follow-up examinations from at least 95% of subjects; however, we will assume conservatively 1732 that the follow-up rate will be 92%. This translates to endpoint data for 408 subjects randomized 1733 to each of the four supplement combinations in the factorial design (1632 in total) and for an 1734 additional 287 females randomized to each of the two vitamin D groups (574 in total). For the 1735 primary comparison of vitamin D versus no vitamin D, we expect 1103 subjects (408 + 408 + 1736 287) per group with follow-up data. For the primary comparison of calcium versus no calcium, 1737 we expect 816 subjects (408 + 408) per group with follow-up data. For the primary comparison 1738 of vitamin D + calcium versus calcium alone, we expect 695 subjects (408 +287) per group with 1739 follow-up data. 1740 1741 Data from our latest trial -- the Aspirin/Folate Polyp Prevention Study -- indicate a three year 1742 adenoma occurrence rate of 45% among placebo subjects (who were asked to avoid NSAID use). 1743 NSAID use by the study subjects may lower the expected adenoma occurrence rate in the 1744 proposed study. Assuming that aspirin confers a 20% decrease in occurrence risk and 33% of

v 20.0 9-11-14 38

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1745 subjects take aspirin daily, we would expect the overall occurrence rate in placebo subjects to be 1746 42%. However, we expect that the subjects who enroll in this study will have lower risk profile 1747 overall because fewer high-risk subjects will be recruited. Thus, for the purpose of power 1748 computations, we assume that the occurrence rate among placebo subjects will be 37%. Power 1749 calculations are based on standard formulae for power of the χ2 test and assume no interaction 1750 between the supplements. Power for the vitamin D effect assumes a calcium relative risk of rc = 1751 0.80, and power for the calcium effect assumes a vitamin D relative risk of rd = 0.75. Table 1 1752 (below) shows the statistical power to detect various differences in occurrence risk for the 1753 primary comparisons of adenoma occurrence rates. The results indicate that we will have good 1754 statistical power to detect relative risks of 0.80 or less for the marginal vitamin D effect and the 1755 marginal calcium effect. We will have adequate power to detect a relative risk of 0.75 or less for 1756 the effect of vitamin D in the presence of calcium. 1757 1758 The detectable relative risk for the vitamin-D-by-calcium interaction effect is approximately 1759 rcd = 0.60 (or 1.67). This was determined based on a z-test comparing log relative risks 1760 assuming adenoma occurrence rates as follows: placebo alone, p; calcium alone, rcp, vitamin D 1761 alone rdp, and vitamin D + calcium, rcdrdrcp, where rc = 0.80, rd = 0.75 and p = 0.37. 1762 Table 1. Statistical power* for primary study comparisons Relative Risk Comparison (expected control occurrence) N per group 0.70 0.75 0.80 0.85 A - Original power calculations: Vitamin D vs. no vitamin D (35%) 905 >0.99 0.98 0.89 0.67 Calcium vs. no calcium (35%) 670 >0.99 0.94 0.79 0.54 Vitamin D + calcium vs. calcium (32%) 570 0.95 0.85 0.67 0.43 B- Updated power calculations: Vitamin D vs. no vitamin D (32%) 1103 > 0.99 0.99 0.92 0.70 Calcium vs. no calcium (32%) 816 > 0.99 0.95 0.82 0.57 Vitamin D + calcium vs. calcium 695 0.97 0.88 0.70 0.46 (30%) *Statistical power is based on a chi-squared test with a 2-sided alpha level of 0.05. Calculations assume a baseline occurrence rate of 37% among placebo subjects and no interaction between the supplements. Power for vitamin D comparisons assumes a relative risk of 0.8 for calcium, and power for calcium comparisons assumes a relative risk of 0.75 for vitamin D. 1763 1764 We will also have good statistical power to detect interaction effects between vitamin D 1765 supplementation and VDR genotype. The total expected sample size of 2206 patients with 1766 follow-up data will be used in these analyses, since all patients will be randomized in the vitamin 1767 D component of the study, and all patients will be assessed for VDR genotype. The statistical 1768 power calculations, given in the table below (Table 2) as the interaction relative risks detectable 1769 with 80% power, assume that VDR genotype is classified as two groups (positive and negative), 1770 the prevalence of the variants ranges from 20% to 50%, and the marginal relative risk for 1771 vitamin D supplementation is 0.75. The calculations also assume that the adenoma occurrence 1772 rate in the control group is 32% and VDR-genotype has no marginal effect on recurrence rates. 1773 For interaction effects between vitamin D levels (above versus below the median) and vitamin D 1774 supplementation, the same computations apply, using the 50% prevalence. 1775

v 20.0 9-11-14 39

Downloaded From: https://jamanetwork.com/ on 09/26/2021 Table 2. Detectable relative risks for the interaction effect of vitamin D supplementation and VDR genotype. Prevalence of VDR genotype Detectable RR 20% 30% 40% 50% Original 0.56 0.62 0.65 0.66 Updated 0.59 0.64 0.66 0.67 1776 1777 12.3 Methods for Randomization and Stratification 1778 Treatment assignments will be randomly generated using a computer program. Separate 1779 assignment lists will be generated for each stratum. Stratification factors are gender, Study 1780 Center, anticipated follow-up interval (3 or 5 years) and whether the patient is participating in 1781 both the vitamin D and calcium components or only the vitamin D component (the latter 1782 component is only for women who wish to take calcium supplements). Treatment assignments 1783 will be made in blocks to ensure equal numbers of patients across the treatment groups at various 1784 time intervals. The size of the blocks will vary in a random fashion. Randomization will occur 1785 only once a patient satisfies all eligibility criteria, including a successful run-in period. 1786 1787 12.4 Outcome Measures 1788 For each subject, we will tabulate the number of mucosal lesions found, and record their size, 1789 histological characteristics, and location in the bowel. Each polyp will be classified (using 1790 standard criteria) as neoplastic (tubular, tubulovillous, villous, or serrated adenomas or cancer), 1791 hyperplastic, hamartomatous, retention, lymphoid, or other. In addition, for the neoplastic 1792 polyps, a statement with regard to the degree of atypia present will be made using standard 1793 criteria. 1794 1795 The main end point of the trial is the occurrence of neoplastic polyps of the large bowel in the 1796 interval between randomization and the anticipated follow-up colonoscopy (3 or 5 years after the 1797 qualifying exam). As in our previous trials, our primary endpoint will be a dichotomous measure 1798 of any occurrence of neoplastic polyps (adenomas). Secondary analyses will focus on the 1799 number and size of neoplastic polyps, the occurrence of advanced adenomas, and effects within 1800 subgroups defined by baseline 25-OH vitamin D levels and by VDR genotype. 1801 1802 12.5 Statistical Analysis 1803 All analyses will be conducted according to the principle of intention to treat. The analysis 1804 population will consist of all randomized patients, with patients being grouped according to 1805 randomized treatment assignment regardless of whether this treatment was actually delivered. 1806 1807 To address our primary hypotheses, the primary statistical analysis will use contingency tables 1808 and standard χ2 tests to compare adenoma occurrence between treatment groups. There will be 1809 three primary comparisons: 1) vitamin D versus no vitamin D, and 2) calcium versus no calcium 1810 and 3) vitamin D + calcium versus calcium alone. Comparison 1 will collapse numbers of 1811 subjects across calcium supplementation groups to assess the marginal effect of vitamin D. 1812 Comparison 2 will collapse numbers of subjects across the vitamin D supplementation groups to 1813 assess the marginal calcium effect and will exclude women who do not consent to randomization 1814 in the calcium component of the study. Comparison 3 will focus only on subjects taking calcium; 1815 no collapsing is necessary. Effects will be summarized using risk ratios rather than odds ratios

v 20.0 9-11-14 40

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1816 to improve interpretation. Two-sided significance levels of 0.05 will be taken to denote 1817 statistical significance. 1818 1819 As in our previous trials, subsequent, more detailed, multivariate analyses will consist of 1820 generalized linear models for binary data to estimate adjusted risk ratios (i.e., using a log link 1821 function) and confidence intervals for neoplastic polyp occurrence among the treatment groups. 1822 We anticipate that the distribution of relevant covariables will be approximately balanced across 1823 the treatment groups because of random assignment in a relatively large sample. Some 1824 imbalances may occur, making it necessary to critically examine possible covariates. These 1825 include number of previous adenomas, size of qualifying adenomas, selected dietary variables 1826 (e.g., calcium intake) or sunlight exposure. The distribution by treatment group of these 1827 covariates (for the whole sample as well as by center) will be assessed. For those variables with 1828 large and systematic imbalances, stratified analyses will be employed to confirm or modify the 1829 preliminary analyses. Factors used to stratify the randomization (center, planned follow-up 1830 interval, full factorial participation) will be included as covariates. We will also use these 1831 models to evaluate the interaction between vitamin D supplementation and calcium 1832 supplementation. The interaction effect will be tested by including the appropriate product term 1833 in the model. This interaction analysis will include only those subjects enrolled in the full 1834 factorial design (i.e., women wishing to take calcium supplements will be excluded). We do not 1835 expect differential treatment effects according to age, gender or race, but we will consider the 1836 corresponding interactions for the treatments. 1837 1838 Analyses pertaining to the secondary hypotheses will use methods similar to those described for 1839 the primary hypotheses. The comparison of calcium + vitamin D versus vitamin D alone 1840 (hypotheses 4) will be dealt with in the same manner as the primary hypotheses. Comparison of 1841 treatment effects within subgroups defined by VDR genotype or baseline vitamin D level will 1842 initially involve contingency tables stratified by genotype or vitamin D level. We will then 1843 apply generalized linear models with appropriate interaction terms and other covariates. If a 1844 possible (i.e., p < 0.10) interaction is observed, we will focus on effects within strata of the 1845 effect-modifying factor. In some analyses we expect to be able to consider the genotype data as 1846 a 3-level variable (homozygous wild type, heterozygous, homozygous variant) since the 1847 prevalence of the least common of these (homozygote variant) is approximately 15% for both the 1848 polymorphisms used. Some analyses, however, will require grouping the heterozygous subjects 1849 with those who are homozygous variant. 1850 1851 In addition to the planned treatment comparisons, we will conduct important subgroup analyses. 1852 For example, we will assess the treatment effects according to self-reported NSAID use during 1853 the trial. We will group subjects as having low, medium and high NSAID use during the study 1854 and add this exposure to our regression models and assess its main effect and its modification of 1855 the vitamin D and calcium risk ratios. In similar analyses, we will assess the impact of baseline 1856 vitamin D levels and VDR polymorphisms as main effects and as modifiers of the intervention 1857 effects. 1858 1859 We will also conduct analyses regarding the number and characteristics of polyps. The number 1860 of adenomas per patient will be evaluated using contingency tables (with groupings such as 0, 1, 1861 2+ adenomas) as well as multivariate Poisson regression with the counts themselves as the 1862 dependent variable. The size of neoplastic polyps will be incorporated using various 1863 dichotomous endpoints; if sufficient adenomas at least a cm in diameter are found, we will use

v 20.0 9-11-14 41

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1864 that cut point. Adenomas with various histological characteristics (serrated, 1865 tubulovillous/villous, advanced dysplasia/cancer) will also be considered as dichotomous 1866 endpoints as outlined above for all adenomas; in particular, we will assess effects on “advanced 1867 histology adenomas” (villous component, advanced dysplasia) or “advanced adenomas 1868 (advanced histology or 1+ cm. in diameter). 1869 1870 Analyses regarding the main hypotheses will not be possible until all subjects have completed 1871 follow-up, after the period covered by this application. We do not anticipate stopping this trial 1872 early for efficacy, since early termination biases estimates of effect,189 and in this population 1873 under colonoscopic surveillance, there is little likelihood of harm from continuing until the 1874 planned study completion. However, for toxicity, we anticipate discussing with the Safety and 1875 Data Monitoring Committee the adoption of stopping guidelines that account for multiple 1876 analyses over time (e.g., using O’Brien-Fleming boundaries).190 Such guidelines have been 1877 established in our Aspirin/Folate Polyp Prevention Study to consider possible treatment effects 1878 on mortality and coronary heart disease. 1879 1880 12.6 Assumptions 1881 Only standard assumptions for statistical analysis will be made. These include: (1) that the study 1882 sample is representative of the eligible patient population; (2) the protocol procedures for 1883 treatment and follow-up of individual patients will result in an unbiased estimate of the treatment 1884 effect; (3) regression models used for the multivariate analyses appropriately fit the empiric data. 1885 Because this study uses a “gold-standard” approach in its design, namely with random treatment 1886 assignment and double blinding of treatment assignment, the possibility of systematic bias is 1887 minimal. 1888 1889 12.7 Compliance and missing data 1890 Missing data may arise due to patients missing follow-up colonoscopies. In past studies by the 1891 Polyp Prevention Group, the percent of missing exams has been small. In addition, because the 1892 treatment assignment is random, and both subjects and investigators are blinded to treatment 1893 assignments, it is not likely that missing exams will generate a bias. Patients with missing 1894 follow-up data will be excluded from the statistical analysis of the main endpoints. 1895 1896 12.8 Interim analyses 1897 No formal interim statistical analysis is planned for this study. The study will not stop early for 1898 efficacy. The study will be monitored by an independent Safety and Data Monitoring 1899 Committee. The Project Coordination Center will provide any data analysis reports the 1900 committee requires. 1901 1902 12.9 Ancillary analyses 1903 Aside from posited effects on colon carcinogenesis, calcium and vitamin D may have a modest 1904 protective effect on cardiovascular disease risk and on fractures. In our routine follow-up, we 1905 will monitor hospital admissions for all cardiovascular events and will thus be able to compare 1906 the event rates in subjects treated with calcium with those given placebo. However, small 1907 numbers of events are expected and our power to detect meaningful differences is likely to be 1908 low. We will also regularly ask our participants to report fractures they have sustained after 1909 randomization. As in our previous calcium trial, we will carefully follow all subjects for cancer 1910 at other sites. 1911

v 20.0 9-11-14 42

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1912 1913 13.0 Ethical and regulatory considerations 1914 1915 13.1 Form FDA 1572 1916 The principal investigator at each Study Center will provide a signed Form 1572 stating that the 1917 study will be conducted in compliance with regulations for clinical investigations as required by 1918 FDA regulations. 1919 1920 13.2 IRB Approval 1921 Prior to initiating the study at any of the participating Study Centers the Principal Investigator at 1922 that center will obtain written approval to conduct the study from the appropriate IRB. In 1923 addition, IRB approval will be required from any recruitment sites where subjects are recruited 1924 that are not covered by the Study Center IRB. Should changes to the study protocol become 1925 necessary, protocol amendments will be promptly submitted in writing to the IRBs and approval 1926 received prior to implementation. Dates of IRB submission(s) and approval(s) and copies of the 1927 approved documents will be submitted to the Project Coordination Center for tracking and 1928 quality control purposes. 1929 1930 13.2.1 Potential Risks to Subjects and Protections Against Risks 1931 The study’s physical risks are those associated with the study agents. Overconsumption of 1932 calcium and Vitamin D may cause conditions such as hypercalcemia, kidney stones, and renal 1933 insufficiency, which are potentially serious. Calcium supplements may interact with some 1934 commonly used drugs. Serious drug interactions between the study tablet and other medications 1935 are unlikely, but possible. In most cases this interaction can be minimized by staggering the 1936 administration of the drug. The likelihood of these complications is very small given the low 1937 doses of the study agents used and the many safety procedures used to protect against this risk. 1938 These protections will include: 1939 • excluding from participation in the trial those individuals that are at increased risk for 1940 these conditions based on their medical history and baseline measurements of blood 1941 levels of calcium, creatinine and vitamin D. 1942 • educating subjects about the symptoms of hypercalcemia. 1943 • monitoring subjects at six month intervals for any adverse symptoms, medical diagnoses 1944 and hospitalizations, or excess dietary or supplemental intake of calcium and vitamin D; 1945 and discontinuation of study medications when indicated due to symptoms, adverse 1946 clinical events, or excess consumption of these agents outside of the study 1947 • asking participants to report all medications being taken during study participation and 1948 advising them to consult with their physician about staggering the administration of 1949 current drugs or new medications that are begun during study participation. 1950 • measuring calcium, creatinine and/or vitamin D levels approximately one year and, 1951 where applicable, three years after randomization; patients who have developed 1952 hypercalcemia, renal insufficiency or very high levels of 25-OH vitamin D (> 100 ng/ml) 1953 will be withdrawn from treatment. 1954 • reviewing adverse events by blinded (and if necessary unblinded) treatment groups by the 1955 Safety and Data Monitoring Committee; this Committee will have overall responsibility 1956 for interpreting data on adverse events and recommending actions to ensure that subjects 1957 are not exposed to undue risks. 1958

v 20.0 9-11-14 43

Downloaded From: https://jamanetwork.com/ on 09/26/2021 1959 A physical risk that may be associated with insufficient intake of calcium or vitamin D over the 1960 long term is an increased risk for osteoporosis, a condition in which increased bone fragility can 1961 lead to fractures. This risk may be greater among post-menopausal women who undergo 1962 accelerated bone loss around the time of the menopause. The risk may be increased if subjects 1963 randomized to placebo reduce their dietary intake of calcium or vitamin D or stop taking 1964 calcium- or vitamin D-containing dietary supplements as a result of their participation in this 1965 trial. To protect against this risk: 1966 • Female subjects will be offered the option of being randomized to calcium alone or to 1967 calcium plus vitamin D. These women will receive 1200 mg of elemental calcium daily 1968 in their study pills. 1969 • Subjects will be cautioned NOT to reduce their dietary intake of calcium or vitamin D 1970 containing foods during their participation in this trial UNLESS we alert them that their 1971 intake is too high. Dietary intake will be assessed with a brief dietary screener at 1972 enrollment and randomization, and at six month intervals after randomization. Subjects 1973 will be counseled that dietary intake of calcium and vitamin D equivalent to about one 1974 quart of milk (about 1200 mg elemental calcium and 400 IU vitamin D) daily is 1975 acceptable for participation in this trial. 1976 • The levels of 25-(OH)-vitamin D in the blood will measured at approximately one year 1977 and, where applicable, three years after randomization and at the end of treatment. 1978 Subjects who have developed low levels of 25-(OH)-vitamin D (<10ng/ml) will be 1979 notified and withdrawn from treatment so that they can take dietary supplements. 1980 1981 Another physical risk is that associated with the study blood draws. The blood drawing 1982 procedure requires that a needle be inserted into an arm vein and blood withdrawn. Almost all 1983 donors experience slight pain at the site of the needle insertion and some may develop a small 1984 bruise. The risk of this discomfort from the blood draw is likely but not serious. 1985 1986 Social risks could occur if confidentiality is violated. Dissemination of information obtained 1987 from research, including genetic analyses, has the potential of adversely affecting employability 1988 or insurability. 1989 To protect subject confidentiality, the following steps will be taken: 1990 • Each subject will be assigned a study identification number and a two letter study 1991 identification code (consisting of the first letter of the subject’s first name and the last 1992 letter of the subject’s last name). All data transfers regarding a subject will be labeled 1993 only with these identification codes. 1994 • The main clinical trial database, which is maintained by the Dartmouth Project 1995 Coordination Center, will contain only the data elements allowed in a ‘limited data set’ as 1996 defined by HIPAA, with the exception of the pathology slide number, which will be used 1997 solely to identify and track receipt and review of these slides by the study pathologist and 1998 their return to the pathology lab of origin. 1999 • The pharmacy database, which is used for the purpose of pill shipments by the Dartmouth 2000 Project Coordination Center and which contains only the participants names, addresses 2001 and telephone numbers, will be kept separate from the main clinical trial database and 2002 will not be directly linked to any other information collected about the participant during 2003 this study. This data will also be used for contacting some participants for the two extra 2004 interviews to evaluate our study methods (e.g. the assessment of blinding effectiveness) 2005 and for mailing Cold and Flu Questionnaires. Finally, study-wide communications (e.g.,

v 20.0 9-11-14 44

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2006 the Study Newsletters) may also be sent by pharmacy personnel from the Project 2007 Coordination Center using this database. 2008 • Medical record forms (endoscopy and pathology reports) will be submitted to the Project 2009 Coordination Center labeled only with coded study IDs. 2010 • Blood samples sent to the Dartmouth Hitchcock Medical Center laboratory will be 2011 labeled and tracked only with unique barcoded numbers. 2012 • Pathology slides generated during routine clinical care are generally labeled only with a 2013 local pathology ID number, however, in some cases other identifying information such as 2014 subject names may also be included as per the local pathology laboratory standard of 2015 practice. Slides sent to the Dartmouth Project Coordination Center and to the Study 2016 Pathologist will be tracked only with the pathology number. These slides will be 2017 returned to the laboratory of origin once they are read by the Study Pathologist, unless 2018 the subject consents to their storage for future research and permission is obtained from 2019 pathology laboratory of origin. 2020 • All computer systems and programs will be equipped with password protection. 2021 • All network-based study communications will be encrypted. 2022 • All printed study materials at the Study Centers and at the Dartmouth Project 2023 Coordination Center that contain identifying information, such as name, address, 2024 telephone number and social security numbers, will be stored in locked filing cabinets. 2025 • All study personnel will be trained to respect and protect subject confidentiality; 2026 information regarding subjects will be discussed only with other study personnel 2027 • We have obtained a Certificate of Confidentiality from the NIH to protect the genetic 2028 information generated by this study 2029 As well, the seriousness of this risk is allayed by the fact that the type of genetic analyses to be 2030 performed in this study are very unlikely to yield any clinically significant information that could 2031 be used to predict future health status. 2032 2033 A potential psychological risk could occur if patients feel a sense of coercion to participate in the 2034 trial. The likelihood is small, since patients will be assured in the informed consent discussion 2035 during the intake process that participation is purely voluntary, and that they may decline to 2036 participate or withdraw from the study at any time with no impact on their regular medical care. 2037 2038 13.2.2 Potential Benefits 2039 Should calcium or vitamin D prove effective in preventing cancer, the public will clearly benefit. 2040 The relative lack of toxicity and simplicity of administration of these agents almost certainly will 2041 make them acceptable to large numbers of asymptomatic people. Such a preventive strategy is 2042 likely to be more acceptable and effective than recommendations to avoid life style habits that 2043 may only modestly increase risk. The potential benefit of a simple, safe, and inexpensive 2044 prophylactic is clear. The actual risks to individuals appear to be minimal while the potential 2045 benefit to the public (and possibly to the study subjects themselves) is great, thus the risk:benefit 2046 ratio favors conducting this study. 2047 2048 The proposed research will have both public health and scientific importance. It has the 2049 potential to confirm other experimental and observational studies indicating that calcium and 2050 vitamin D supplementation protect against colorectal neoplasms. It will show, for the first time, 2051 whether both agents together are more effective than calcium alone. From a public health 2052 perspective, a positive finding will suggest a low-cost strategy for reducing the burden of

v 20.0 9-11-14 45

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2053 colorectal cancer in the general population. From a scientific perspective, the study will permit 2054 investigation of the mechanisms underlying the beneficial effects of the study agents, and thus 2055 elucidate the pathogenesis of cancer in the large bowel. 2056 2057 13.3 Informed Consent 2058 The informed consent process will begin during the first patient contact and continue throughout 2059 the study. The Principal Investigator at each Study Center may delegate the responsibility of 2060 obtaining informed consent to the center’s study coordinator. During the initial in-person intake 2061 appointment, the study coordinator will conduct an informed consent discussion, focusing on the 2062 research nature of the study, its purposes, expected duration of participation, study procedures 2063 including randomization to active agent or placebo, possible risks or discomforts, possible 2064 benefits, and confidentiality. It will stress the voluntary nature of participation and the right to 2065 withdraw. Willing patients will document their consent to participate by signing and dating an 2066 informed consent document presenting this information in written form and providing 2067 authorization to access personal health information for HIPAA compliance. The discussion will 2068 take place at the Study Center in a private, comfortable environment free of interruptions and 2069 with sufficient time allocated to address all questions and concerns. Subjects are given a copy of 2070 the signed and dated informed consent documents; the original signed and dated consents are 2071 placed in the subject’s study file, and a copy is also placed in the medical record. 2072 2073 13.4 Data and Safety Monitoring Plan 2074 A Data and Safety Monitoring Committee will regularly review interim data to assess 2075 compliance, monitor toxicity, and recommend whether the trial should continue. Committee 2076 members are independent experts chosen on the basis of their expertise and scientific rigor. 2077 They are not associated with the trial or with the pharmaceutical companies that supply the study 2078 agents. The Principal Investigator, together with the study’s Steering Committee, carefully 2079 assess the background of each member to rule out the possibility of conflicts of interest with or 2080 financial interests in the research outcome. Committee members’ areas of expertise span the 2081 disciplines relevant to the conduct of GI clinical trials, including epidemiology, clinical trials, 2082 pharmacology, biostatistics, and clinical care of adenoma patients. 2083 2084 The Committee will confer at six-month intervals, generally by telephone conference call, with 2085 in-person meetings, as needed. The study’s Principal Investigator, John Baron, MD, and the 2086 study’s Biostatistician as well as other Project Coordination Center investigators as needed, will 2087 participate in the meetings’ open sessions. If needed, the Committee’s voting members may 2088 then choose to discuss in closed session unblinded data or other material that should be kept 2089 confidential from the investigators. Under the supervision of the study Biostatistician, the lead 2090 programmer at the Project Coordination Center will prepare a bi-annual “Report to the Data and 2091 Safety Monitoring Committee” which includes interim outcome data. The study Biostatistician 2092 and programmer ensure that the full report is kept strictly confidential. 2093 2094 The Committee will have the responsibility to review the research protocol and to evaluate the 2095 progress of the trial overall and at each participating Study Center. This will include accrual, 2096 compliance with pill-taking and study exams, use of calcium and Vitamin D supplements, 2097 prescription drug use, mortality, episodes of hospitalization, toxicities, adverse events, disease 2098 diagnoses, reported changes in health, protocol violations and data quality. Serious unexpected 2099 events will be disclosed to the committee in between meetings. The Committee will also review 2100 interim endpoint data. The Committee will evaluate participant risk vs benefit as the trial

v 20.0 9-11-14 46

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2101 progresses, considering evolving scientific discoveries or treatment options that may affect the 2102 desirability of continued treatment. At the conclusion of each meeting, the Committee will 2103 recommend whether the trial be continued. Following each meeting of the Data and Safety 2104 Monitoring Committee, its secretary will prepare a report on the questions raised by Committee 2105 members, monitoring recommendations, and recommendations for the continuation of the trial. 2106 This report will be distributed confidentially to meeting participants. The Committee’s secretary 2107 will also prepare a redacted summary of this report, focusing on safety issues, for distribution to 2108 Study Center co-investigators and their IRBs. 2109 2110 13.5 Project Coordination Center, Sponsor or FDA monitoring 2111 The Dartmouth Project Coordination Center, the FDA and the NCI or their designees may 2112 monitor/audit various aspects of the study. These monitors will be given access to facilities, 2113 supplies and records to review and verify data pertinent to this study. 2114 2115 2116 13.6 Record Retention 2117 Clinical records for all subjects studied will be maintained by the Investigator in a secure storage 2118 facility and stored until the Dartmouth Project Coordination Center directs the material to be 2119 destroyed. Once study enrollment is complete and summary statistics have been analyzed, the 2120 Project Coordination Center will notify all Study Centers that they may destroy all data collected 2121 from screened subjects who were not subsequently enrolled into the study. 2122 2123 13.7 Certificate of Confidentiality 2124 To help protect subject privacy, we have obtained a "Certificate of Confidentiality" from the 2125 National Institutes of Health. With this Certificate we cannot be forced to disclose information 2126 that may identify a subject, even by a court subpoena, in any federal, state, or local civil, 2127 criminal, administrative, legislative, or other proceedings. Although the Certificate adds another 2128 layer of protection, it does not apply in the following situations: 1) When we receive requests 2129 for information that come from the United States Government for the purpose of evaluating or 2130 auditing research projects funded by the Federal Government, or for information needed to meet 2131 the requirements of the Food and Drug Administration (FDA); and 2) If a subject chooses to 2132 give written consent to release research information to an insurer, employer, or other person the 2133 Certificate does not allow us to withhold that information. 2134 2135 14.0 Ancillary Studies 2136 2137 14.1 Calcium/Vitamin D, Biomarkers, and Colon Polyp Prevention (PPS4B) 2138 This “biomarker study” is funded the National Cancer Institute and the Franklin Foundation 2139 through Emory University and the Polyp Prevention Study Group consortium as an adjunct study 2140 to the Vitamin D/Calcium Polyp Prevention Study (parent study). Subjects in the parent study 2141 will be invited to participate in this adjunct study and, if they agree, will be consented separately 2142 to this study. 2143 2144 14.1.1 Objectives 2145 The primary aims are to investigate the effect of calcium and/or vitamin D on the individual 2146 components and aggregate profile of a molecular phenotype panel of biomarkers of risk for 2147 colorectal cancer, and to assess whether this modulation is associated with decreased recurrence

v 20.0 9-11-14 47

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2148 of sporadic colorectal adenomatous polyps. The following biomarkers will be measured in 2149 biopsies of normal-appearing colorectal mucosa: 2150 • inflammation (COX-2 expression) 2151 • early colon carcinogenesis pathway genes (APC pathway: expression of APC, β-catenin, 2152 E-cadherin; mismatch repair pathway: expression of MLH1, bax) 2153 • cell cycle events (long-term proliferation: expression of telomerase; apoptosis inhibition 2154 and promotion: expression of bcl-2, bax) 2155 • autocrine/paracrine growth promotion and inhibition factors (expression of TGFα, 2156 TGFβ1) 2157 2158 The secondary aims are as follows: 2159 • To clarify whether the differences in the biomarkers among the treatment groups at the 3- 2160 or 5-year colonoscopies precede and may predict adenoma recurrence, “non-prep” rectal 2161 biopsies will be taken at baseline and at a 1-year follow-up visit. 2162 • To clarify whether the differences in the biomarkers in rectal mucosa among the 2163 treatment groups at the 3- or 5-year colonoscopies also occur more proximally in the 2164 colon, at the 3- or 5-year colonoscopy—in addition to the rectal biopsies—biopsies will 2165 also be taken from the mid-sigmoid and proximal ascending colon. 2166 • To clarify a) whether the differences in the biomarkers in rectal mucosa among the 2167 treatment groups at the 3- or 5-year colonoscopies are affected by colonoscopic 2168 preparation, and b) whether changes in biomarkers from baseline to one year are 2169 maintained, accumulative, or attenuated by 3 or 5 years, non-prep rectal biopsies will be 2170 taken 7 – 21 days prior to their colonoscopies with biopsies. 2171 • Estimate the normal variability of the biomarker panel in sporadic adenoma patients over 2172 time. This will be done by estimating the inter- and intra-subject components of 2173 variability over the 3 or 5 years. 2174 • Investigate whether biomarker responses to treatments vary according to nonsteroidal 2175 anti-inflammatory drug (NSAID) use or vitamin D receptor (VDR) genotype. 2176 2177 14.1.2 Recruitment and Enrollment Procedures 2178 The parent study defines the pool of potential subjects for the biomarker study. Two additional 2179 exclusion criteria for this adjunct study are: a) unable to be off aspirin for 7 days, and b) a 2180 history of bleeding disorder or current use of anticoagulant medication. Subjects will be invited 2181 to participate in the Biomarker Study in one of two ways: 2182 2183 1) Rectal biopsies at 3- or 5-year follow-up colonoscopy only: Randomized subjects from all 2184 Study Centers participating in the parent study will be invited to participate in rectal 2185 biopsies that occur at their 3- or 5-year follow-up colonoscopy. Of the approximately 2186 2,400 randomized participants in the parent study, we expect to recruit approximately 2187 1,328 to participate in this portion of the biomarker study. 2188 2189 Study coordinators will contact subjects by mail or telephone several months prior to their 2190 scheduled 3- or 5-year follow-up colonoscopy to describe the biomarker study and to invite 2191 the subject to participate. Interested subjects will be consented to the biomarker study at an 2192 in-person visit prior to their 3- or 5-year follow-up colonoscopy. 2193

v 20.0 9-11-14 48

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2194 2) Additional Biopsies: Subjects from the South Carolina and Emory Study Centers only who 2195 have been enrolled into the parent study, but not yet randomized, will be invited to 2196 participate in the following additional biopsies: 2197 a. Biopsies at outpatient sigmoidoscopy: rectal biopsies will be collected during 2198 outpatient sigmoidoscopies occurring at randomization, at the 1-year follow-up 2199 visit, and at a visit 7 – 21 days before the 3- or 5-year follow-up colonoscopy 2200 b. Additional biopsies at follow-up colonoscopy: biopsies will be taken from the 2201 mid-sigmoid and proximal ascending colon at the 3- or 5-year follow-up 2202 colonoscopy (in addition to the rectal biopsies collected at this follow-up 2203 colonoscopy). 2204 2205 We expect to recruit approximately 200 subjects to participate in this portion of the 2206 biomarker study. Study coordinators will contact subjects by telephone prior to their 2207 deadline for randomization in the parent study to describe the Biomarker Study and to 2208 invite the subject to participate. Interested subjects will be consented to the Biomarker 2209 study at an in-person visit at the time of their randomization into the parent study. 2210 2211 14.1.3 Biopsy Collection and Analysis 2212 All colorectal biopsies will be obtained using currently successful protocols in the procedure 2213 rooms used by the participating gastroenterology offices by a physician trained in the procedure, 2214 with the assistance of the local Study Coordinator. For randomized participants from all Study 2215 Centers who consent to this adjunct study, rectal biopsies only will be obtained only during the 2216 patient’s follow-up colonoscopy. For enrolled participants from South Carolina and Emory who 2217 consent to the additional biopsies, outpatient rectal biopsies will be obtained via sigmoidoscopy 2218 at randomization, one-year follow-up, and 7 – 21 days prior to their clinical colonoscopy; also, in 2219 addition to rectal biopsies, biopsies will be obtained from the mid-sigmoid and proximal 2220 ascending colon during the patient’s follow-up colonoscopy. 2221 2222 Prior to the colonoscopies, participants will use a PEG (polyethylene glycol based) preparation 2223 in order to clear out the bowel. This specific type of preparation is used because it does not 2224 interfere with the biomarker measurements that will be performed. The PEG preparation will be 2225 provided by the study if it is not available as a standard preparation at the local gastroenterology 2226 clinic. No bowel-cleansing preparation is used for the outpatient rectal biopsies. 2227 2228 At least six adequate rectal biopsies must be obtained during the colonoscopy (from all 2229 participants) or sigmoidoscopy (from participants consenting to the additional biopsies). In 2230 addition, at least three adequate colonic biopsies must be obtained from each of the mid-sigmoid 2231 and proximal ascending colon sites during the follow-up colonoscopy for participants consenting 2232 to the additional biopsies. If a biopsy pinch is inadequate, another will be obtained. Biopsy 2233 specimens are immediately examined and processed for storage in formalin. All biopsy samples 2234 will be sent to Dr. Roberd Bostick’s laboratory at Emory University for analysis. The 2235 biomarkers will be measured by immunohistochemical methods followed by quantitative image 2236 analysis of the total expression and distribution of the expression of the biomarkers in colon 2237 crypts. 2238 2239 14.1.4 Sample Size 2240 Adenoma recurrence in the parent study is expected to be similar to that found in the Calcium 2241 Polyp Prevention Study, in which, among the 832 subjects who completed the study, 52% of the

v 20.0 9-11-14 49

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2242 placebo and 45% of the calcium group had a recurrence of ≥ 1 adenoma, for a statistically 2243 significant relative reduction in adenoma recurrence of 15% (adjusted RR = 0.85; 95% CI 0.74 – 2244 0.98). We expect to recruit 1,328 to participate in the rectal biopsies at colonoscopy (from all 2245 study centers) and we expect 200 of these subjects (from Emory and South Carolina) to 2246 participate in the additional biopsies as well. 2247 2248 Primary Aims: For the estimated power for Primary Aim 1a, detection of an effect of treatment 2249 on the biomarker level, we assumed testing will be performed using an F-test based on a general 2250 linear model for each treatment separately, with 2-sided testing at α = 0.05, and that the 2251 biomarkers are log normally distributed. For each of the biomarkers we will have greater than 2252 80% power for testing each of the hypotheses described in the specific aims: ie, calcium and/or 2253 vitamin D will change the total expression of each of the individual biomarkers, and these 2254 changes will be associated with decreased adenoma recurrence. For most of the biomarkers, we 2255 will have over 80% power to detect a treatment effect even if the effect is only half as large as 2256 differences observed in a previous case-control study. In addition, the power to detect a 2257 calcium-vitamin D interaction for effect sizes (in SDs) of 0.08, 0.12, 0.16, 0.24, and 0.24 is 24%, 2258 47%, 71%, 88%, and 96%, respectively. 2259 For the estimated power for Primary Aim 1b, detection of an association between each 2260 biomarker level and recurrence of one or more adenomas, we estimated power by assuming 2261 testing will be performed using a logistic model, with 2-sided testing at α = 0.05, 50% polyp 2262 recurrence at the mean biomarker level, marker-covariate R2s = 0.2, and assuming that the 2263 marker is log normally distributed. For each of the biomarkers we will have > 80% power for 2264 testing most of the hypotheses described in the specific aims. 2265 2266 Secondary Aims: The sample size for the secondary aims (n = 200; 50/treatment group) is 2267 unlikely to provide sufficient power to allow definitive statistical testing of the hypotheses for 2268 these ancillary aims. However, the sample size will be sufficient for refining our biomarker 2269 profile and sample size estimates for the primary aims, which will mostly be addressed during a 2270 subsequent competitive grant renewal period. The sample size will also be sufficient for the 2271 other purpose of the first four secondary aims of the biomarker study, which is to provide enough 2272 supplemental, descriptive data to support a qualitative assessment of the quality and 2273 generalizability of the data and results obtained from the primary aims of the biomarker study. 2274 The data generated by addressing the secondary aims accomplishes this purpose by providing 2275 assurance that biomarker differences at follow-up colonoscopies in fact are changes that precede 2276 adenoma recurrence, areas of the colon proximal to the rectum are affected by treatment, and 2277 biomarker findings taken during colonoscopy reflect those that are taken without preceding 2278 colon preparation. 2279 2280 For Secondary Aim 1 we calculated the estimated power to detect an association between 2281 recurrence at 3 or 5 years and change in the biomarker from baseline to one year, adjusted for the 2282 baseline biomarker level. We assume 200 subjects (50/treatment group); that a logistic model 2283 will be used with recurrence (yes/no) at 3-5 years as the dependent variable; that the OR per unit 2284 increase in ∆M is 2.0 after adjustment for the baseline biomarker level (where ∆M is the change 2285 from baseline to one year); that the ∆M is correlated with the other independent variables, 2286 baseline biomarker level, treatment group, and time until the follow-up colonoscopy, with R2 = 2287 0.2; and that, among those with average values of the independent variables, adenoma recurrence 2288 at 3-5 years is 50%. With these assumptions, the power to detect an adjusted OR of 0.5 or 2.0 is

v 20.0 9-11-14 50

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2289 about 99% (at R2 = 0.5, the power is 0.93). For Secondary Aim 2, we calculated the estimated 2290 power to detect an effect of treatment on the biomarker level measured in the proximal colon, 2291 just as for the first primary aim, but for effect sizes that were larger. For effect sizes as large as 2292 0.48 SDs, the power to detect such a difference would be about 92%. 2293 2294 14.1.5 Additional Risks 2295 The additional physical risks associated with the adjunct study are those associated with the 2296 collection of the colorectal biopsies which are outside of the standard of care. The procurement 2297 of 1 mm thick colorectal mucosal biopsies is considered a relatively minor procedure with very 2298 low risk. 2299 2300 1) Biopsies at colonoscopy - On all participants at all clinical centers, small pinches 2301 of tissue (biopsies) 1 mm thick (less than 1/16 of an inch) will be taken from the 2302 lower colon, or rectum about 3 – 4 inches in from the anus on normal-appearing 2303 colorectal tissue. A total of 6 – 8 pinches of tissue will be taken. It is anticipated that 2304 these additional biopsies will add about 3 - 5 minutes to the colonoscopy time. 2305 2306 On all participants who agree to the additional biopsies (only at the South Carolina 2307 and Emory centers), 3 – 4 pinches of tissue will also be taken at the mid-sigmoid 2308 colon and at the proximal ascending colon, adding another 3 – 5 minutes to the 2309 colonoscopy time (ie, there will be a total of 12 – 16 biopsies from all three colon 2310 sites combined, adding about 6 – 10 minutes to the colonoscopy time). 2311 2312 The biopsies are a standard clinical diagnostic procedure with very little risk. 2313 Patients do not experience any pain from the biopsies themselves; the only discomfort 2314 will be that from the colonoscopy itself. Almost all patients experience some 2315 cramping during and after a colonoscopy; although unlikely, it is possible that the 2316 biopsies might mildly contribute to the cramping. A possible complication of the 2317 biopsies is minor bleeding. Other complications could be more serious bleeding, 2318 perforation of (that is, producing a hole in) the bowel wall, or infection, which could 2319 result in needing surgery. These complications are extremely rare, so rare that none 2320 have occurred in studies like this one involving over 12,000 persons so far. 2321 2322 2) Outpatient rectal biopsies at sigmoidoscopy – On all participants who agree to the 2323 additional biopsies (only at the South Carolina and Emory clinical centers), outpatient, non- 2324 prep rectal biopsies will be taken via sigmoidoscopy. These sigmoidoscopic biopsies involve 2325 inserting a tube—about as long and as big around as a doctor’s examining finger—through 2326 the anus into the rectum or lower colon to a depth of about 3-4 inches. At this spot, 4 - 6 tiny 2327 pinches of tissue one mm thick (less than 1/16 of an inch) will be taken. The procedure takes 2328 less than two minutes, is painless (the only discomfort is like that of having a rectal exam), 2329 and is very low risk—about like having blood drawn. 2330 2331 Protection against additional risk: All subjects are counseled to refrain from aspirin intake for 7 2332 days prior to biopsy procedures to reduce the risk of bleeding. In addition, potential participants 2333 are screened for contraindications to colorectal biopsies (unable to be off aspirin for 7 days OR a 2334 history of bleeding disorder or current use of anticoagulant medication) and are not enrolled if 2335 these conditions exist. The Data and Safety Monitoring Committee for the parent trial will serve

v 20.0 9-11-14 51

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2336 in this capacity for the adjunct study, by monitoring for potential biopsy complications and 2337 recommending whether the trial should continue. 2338 2339 14.2 Vitamin D/Calcium Mammography Study 2340 The “Vitamin D/Calcium Mammography Study” is an ancillary study to the Vitamin D/Calcium 2341 Polyp Prevention Study (parent study) under the direction of Dr. Susan Gapstur previously at 2342 Northwestern University. Female subjects in the parent study will be invited to participate in 2343 this ancillary study and, if they agree, will be consented separately for this study. 2344 2345 14.2.1 Background and rationale 2346 The radiographic appearance of the breast varies according to differences in the relative 2347 distributions of fat and fibroglandular tissues. The extent of mammographically detected 2348 fibroglandular breast tissue (i.e., percent breast density) is a strong breast cancer risk factor. 2349 Although a large body of evidence shows consistent associations of percent breast density with 2350 age, reproductive factors, body mass index (BMI) and use of postmenopausal hormone 2351 replacement therapy (HRT), there are few data on associations of modifiable lifestyle factors 2352 with breast density. Results of some observational epidemiologic studies suggest that dietary 2353 vitamin D and calcium intake might be associated with a lower risk of breast cancer. In addition, 2354 other observational studies have shown inverse associations of vitamin D and calcium intake 2355 with percent breast density. It is possible that a protective effect of vitamin D on breast cancer 2356 etiology is mediated through vitamin D signaling effects on mammary epithelial cell 2357 differentiation, proliferation and apoptosis. Similarly, calcium also appears to exert anti- 2358 neoplastic effects. 2359 2360 The Vitamin D/Calcium Mammography Study is designed to assess the effects of supplemental 2361 vitamin D and calcium carbonate (and their synergistic effects) on mammographic breast density 2362 as an ancillary study of the Vitamin D/Calcium Polyp Prevention Trial. This multi-centered, 2363 randomized clinical trial provides an ideal opportunity to study the associations of vitamin 2364 D/calcium with breast density for several reasons. First, the primary exposures will be known 2365 quantities of vitamin D and calcium, thus removing the potential measurement errors introduced 2366 by dietary data collection. Second, we will be able to compare changes in density over time 2367 across the calcium only, vitamin D only, both or placebo groups. Third, the availability of high- 2368 quality data collected on potential confounding factors (e.g, hormone use, BMI, menopausal 2369 status, and age) will allow us to control for potential confounding factors that might differ 2370 between groups. Fourth, we are able to explore whether variants in genes in the vitamin D 2371 receptor might modify associations of supplemental vitamin D with percent breast density. If 2372 successful, the results of this study could be used to inform the design of a chemoprevention trial 2373 of vitamin D and/or calcium supplement to prevent breast cancer with percent breast density as 2374 the intermediate endpoint. 2375 2376 14.2.2 Objectives 2377 The primary aim of this study is to assess the effects of supplemental vitamin D3 (1000 IU/daily) 2378 and calcium carbonate (1200 mg elemental calcium/day) on mammographic breast density. 2379 2380 Our primary hypotheses are: 2381 1. Vitamin D3 (1000 IU/day) supplementation reduces mammographic breast density. 2382 2. Calcium carbonate (1200 mg elemental calcium/day) supplementation reduces 2383 mammographic breast density.

v 20.0 9-11-14 52

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2384 Our secondary hypothesis is: 2385 1. The combination of both vitamin D3 (1000 IU/day) and calcium carbonate (1200 mg 2386 elemental calcium/day) supplementation reduces mammographic breast density more than 2387 supplementation with calcium carbonate or vitamin D alone. 2388 An exploratory hypothesis is: 2389 1. The effect of supplementation with vitamin D3 on mammographic breast density will be 2390 modified by polymorphisms at the 3' end of the vitamin D receptor gene. 2391 2. The combination of both vitamin D3 (1000 IU/day) and calcium carbonate (1200 mg 2392 elemental calcium/day) supplementation reduces mammographic breast density compared to 2393 placebo. 2394 2395 14.2.3 Recruitment and Enrollment Procedures 2396 The parent study defines the pool of potential subjects for this study. Two additional exclusion 2397 criteria for this ancillary study are: a) a history of breast cancer, and b) not anticipating having a 2398 mammogram during the duration of the parent study. Subjects will be invited to participate in 2399 the Mammography Study in one of two ways: 2400 2401 1) Women currently being enrolled in the parent study will be invited to participate at the 2402 time of their in-person intake visit. Study coordinators will ask women to complete the 2403 mammography eligibility and intake questionnaire. Those who are eligible will be 2404 invited to participate in this ancillary study. If they are interested, the informed consent 2405 process will be carried out at this visit and documented on the informed consent form. 2406 The subject will also be asked to sign a medical release form to allow collection of their 2407 mammograms. Upon the subject’s randomization in the parent study, the study 2408 coordinator will collect the most recent screening mammogram from prior to enrollment, 2409 as well as subsequent mammograms from at least one year after enrollment into the 2410 parent study up until the end of the subject’s participation in the parent study. Subjects 2411 that consent to this ancillary study but become run-in failures in the parent study will be 2412 dropped from this study and their mammograms will not be collected. 2413 2414 2) Women already randomized into the parent study, will also be invited to participate. 2415 Study coordinators will ask women who are currently randomized in the study if they are 2416 interested in participating in the Mammography Study during an interim study visit (i.e. the 2417 year 1 blood draw), or during phone contact for an interval questionnaire. For interested 2418 and eligible subjects, local IRB procedures for obtaining informed consent will be 2419 followed. Where permissible, the informed consent process will be carried out over the 2420 phone and then documented by mailing an informed consent form and a release of medical 2421 information to the subject for their signature. Once the signed consent form is returned the 2422 study coordinators will collect the most recent screening mammogram from prior to 2423 enrollment, as well as subsequent mammograms from at least one year after enrollment in 2424 the parent study up until the end of the subject’s participation in the parent study. 2425 2426 14.2.4 Collecting Mammogram Information 2427 Once a subject is enrolled in this ancillary study, the study coordinators will ask each woman a 2428 few questions regarding their screening mammography history, including information on where 2429 and when the most recent mammogram was conducted and whether she was taking medications 2430 containing female hormones, such as hormone replacement therapy or birth control, at the time 2431 the mammogram was performed. She will be asked to sign a release of medical information

v 20.0 9-11-14 53

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2432 form to allow collection of one of the images taken at that mammogram. Similarly, at the time 2433 of the interval (semiannual) interview, women enrolled in this ancillary study will be asked if 2434 they had a recent screening mammogram and, if so, where and when that mammogram was 2435 performed. 2436 2437 At the time of a screening mammogram, four images are generally taken: craniocaudal and 2438 medio-lateral oblique images of each the right and left breast. For this study, we will request the 2439 left breast craniocaudal image if available. If that image is not available, we will request the 2440 right breast craniocaudal image. 2441 2442 At each study center, the study coordinators will request the screening mammogram from the 2443 appropriate clinics. It is likely that images from the screening mammogram might be obtained in 2444 one of three ways. First, if available, it would be preferable to have the image from a full-field 2445 digital mammography system. In this case, the image can be electronically transmitted to 2446 investigators at Northwestern University for the assessment of percent breast density. A second 2447 possibility is to have the mammogram digitized at the original institution and to transmit the 2448 digital image to Northwestern University. Using either of these two methods, it is possible to 2449 maintain confidentially by using only the parent study ID number and date of the mammogram 2450 to identify the image. The third possibility is that only a film screen mammogram is available 2451 from an institution that cannot digitize it. In this case, the study coordinator will place a label 2452 with the study ID over any identifying information (e.g., name) on the mammogram. The film 2453 mammogram will then be sent to Northwestern University where it will be scanned. The film 2454 will then be returned to the institution of origin. 2455 2456 At Northwestern University, a trained technician blinded to treatment status will determine the 2457 percent of breast fibroglandular density measured from the craniocaudal image. An automated 2458 analysis will be used to measure the quantity of fibroglandular tissue in the breast. This method 2459 consists of four steps for each digital image: 1) display of the digital mammogram, 2) 2460 determining the area of the entire breast (excluding lesion markers, nipple markers, and 2461 pectoralis major muscle along the chest wall), 3) determining a threshold signal value that 2462 determines the edges of fibroglandular regions, and 4) calculating the breast area (BA) and the 2463 fibroglandular tissue area (FA) within the breast area by summing the number of pixels within 2464 each region. Percent breast density, which is simply the ratio FA/BA x 100, will be calculated. 2465 Percent density provides a 2-dimensional quantitative estimate of breast density that has been 2466 shown to have high inter- and intra-reader reliability in selecting the threshold value and 2467 determining breast density in screen film mammography (SFM). 2468 2469 14.2.5 Statistical Considerations 2470 Study Design 2471 The goal of the parent study is to enroll 3000 total participants and to randomize 2400. Based on 2472 current recruitment rates, approximately 40% of the participants are women; therefore it is 2473 estimated that approximately 960 participants will be women. Of the women recruited we 2474 expect that 624 will be enrolled in the two-arm and 336 will be enrolled in the full-factorial 2475 study. Based on preliminary data that were collected from four study centers (NC, SC, GA and 2476 TX), it is estimated that at least 80% of women randomized will be eligible (i.e., no history of 2477 breast cancer) and will agree to participate in the mammography study. 2478

v 20.0 9-11-14 54

Downloaded From: https://jamanetwork.com/ on 09/26/2021 Vit D and Vitamin D Calcium Placebo TOTAL Calcium Two-arm 312 312 624 Full-factorial 84 84 84 84 336 Total randomized 84 396 396 84 960 No. of eligible women who agree 67 317 317 67 768 to participate. 2479 2480 2481 The design for this ancillary study is a 2 x 2 factorial design as follows: 2482 Calcium - No Calcium – Yes TOTAL Vitamin D – No 67 317 384 Vitamin D - Yes 67 317 384 TOTAL 134 634 768 2483 2484 Statistical Analysis 2485 In preliminary analysis, we will ascertain the distribution of percent breast density to determine 2486 whether there are outliers or evidence of non-normality. If non-normality is indicated within any 2487 of the four study groups, then the density data will be log transformed before statistical analysis. 2488 Group differences in potential confounding variables are likely to be small due to the 2489 randomized nature of the study. However, we will examine descriptive statistics by treatment 2490 group for variables collected at baseline and at each follow-up visit, including demographics, 2491 hormone use, menopausal status, and BMI. We will compute means and standard deviations for 2492 continuous variables, medians and interquartile ranges for ordinal variables, and percentages by 2493 category for categorical variables. Potential group differences will be compared using analysis 2494 of variance (ANOVA) for continuous variables and chi-square tests for dichotomous or 2495 polychotomous variables. If group differences are observed, they will be included as covariates 2496 in analyses. 2497 2498 In this ancillary study, we are collecting the most recent mammogram prior to randomization as 2499 well as each mammogram post-randomization after being on study for at least one year and 2500 through the end of the study. The time from the pre-randomization mammogram to the first 2501 mammogram post-randomization is likely to vary among women. Therefore, to maximize a 2502 potential treatment effect, we will base our primary analysis on the mammogram where women 2503 have been on treatment for the longest period of time (i.e., a post-test comparison). 2504 2505 The primary method of statistical analysis for this ancillary study will be two-factor ANOVA or 2506 analysis of covariance (ANCOVA), to take advantage of the factorial nature of the design. This 2507 analysis will include a factor for Vitamin D (No, Yes), a factor for Calcium (No, Yes) and a 2508 Vitamin D x Calcium interaction term. The presence of a significant interaction will be assessed 2509 first and if the interaction term is statistically significant, and if this is due to the mean density 2510 for the combined Vitamin D + Calcium group being lower than the other three groups, then the 2511 secondary hypothesis will be supported and will show that both supplements synergistically 2512 reduce density more than each supplement alone. If the interaction term is not significant, then 2513 the marginal means in the factorial design will be tested using the main effect for vitamin D to

v 20.0 9-11-14 55

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2514 test hypothesis 1, and the main effect for calcium to test hypothesis 2. (If the interaction term is 2515 significant, then main effects will still be tested, recognizing that there are differential effects). 2516 If group differences are observed, we will then conduct exploratory analyses to determine 2517 whether supplementation leads to a change in breast density (i.e., between pre-randomization 2518 mammogram and post-randomization mammogram). In addition, in exploratory analysis we will 2519 also use ANOVA to test the hypothesis that mammographic breast density is significantly lower 2520 in women randomized to the combination of vitamin D plus calcium compared to the placebo 2521 group. 2522 2523 We will also use ANOVA to test the exploratory hypothesis that the effect of supplementation 2524 with vitamin D on mammographic breast density is modified by polymorphisms at the 3' end of 2525 the vitamin D receptor gene. For this analysis, we will include a dichotomous variable to 2526 represent the presence or absence of the polymorphism, a dichotomous variable for vitamin D 2527 supplement (yes vs. no), and a cross-product term of these two variable. We will also include a 2528 dichotomous variable for calcium (yes vs. no) is in the above analyses calcium is found to be 2529 significantly associated with breast density. 2530 2531 Since all statistical tests are based on a priori hypotheses and are done within the context of the 2532 ANOVA or ANVOCA models, there will be no adjustment for multiple testing in either the 2533 analysis or sample size calculations. 2534 2535 Power/Sample Size Considerations. 2536 Using a two-tailed test with an α=0.05, and 80% power, we computed the minimal detectable 2537 difference in mean percent breast density for each of the comparisons as described for each of 2538 the primary and secondary aims below. 2539 2540 Primary Aim 1: Our first primary aim is to determine if supplementation with vitamin D reduces 2541 mammographic breast density. For this comparison of vitamin D versus no vitamin D we expect 2542 384 (317 + 67) women per comparison group. This sample size will have 80% power to detect 2543 an effect size of 0.20, where effect size is defined as the mean difference in mammographic 2544 density between the vitamin D and no vitamin D groups, divided by the within group standard 2545 deviation (assumed to be the same for both groups). The units for the mean and standard 2546 deviation are percent breast density denoted by ‘%’. In a large cross-sectional study of primarily 2547 Caucasian women aged 40-93 years, percent breast density was determined by analyzing 2548 mammograms (Vachon et al, 2002). In that study, a standard deviation of 15% was observed. 2549 Given the similar age range of women in that study and our proposed study, we based our power 2550 estimates assuming a standard deviation of 15%. Therefore, the minimal detectable difference 2551 between the vitamin D groups is 3% (=.20 x 15). 2552 2553 Primary Aim 2: Our second primary aim is to determine if supplementation with calcium 2554 carbonate (calcium) reduces mammographic breast density. For this comparison of calcium 2555 versus no calcium, we expect 634 (317 + 317) women in the calcium group and 134 women (67 2556 + 67) in the no calcium group. This sample size will have 80% power to detect an effect size of 2557 0.27. Assuming a standard deviation of 15% for percent breast density, the detectable difference 2558 between the calcium groups is 4.0% (=.27 x 15). 2559

v 20.0 9-11-14 56

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2560 Secondary Aim 1: Our secondary aim is to determine if supplementation with both vitamin D 2561 and calcium reduces mammographic breast density more than supplementation with calcium 2562 alone or with vitamin D alone. This vitamin D by calcium interaction reflects the vitamin D 2563 effect in the absence of calcium (67 vs 67 women) compared with the vitamin D effect in the 2564 presence of calcium (317 vs 317 women). These sample sizes will have 80% power to detect an 2565 effect size of 0.54. Here, effect size is defined as (vitamin D effect in the absence of calcium – 2566 vitamin D effect in the presence of calcium)/within group standard deviation (grouping is the 4 2567 treatment combination groups and the standard deviation is assumed to be the same for the 4 2568 groups). Assuming a standard deviation of 15% for percent breast density, there is 80% power 2569 to detect a vitamin D vs no vitamin D mean difference in the presence of calcium that is 8% 2570 higher than the vitamin D vs no vitamin D mean difference in the absence of calcium. For 2571 example, if the mean percent density in the 67 women not on vitamin D and not on calcium is 2572 30%, and the mean percent density in the 67 women on vitamin D and not on calcium is 27% 2573 (vitamin D effect in the absence of calcium is 30 – 27 = 3%), and if the mean percent density in 2574 the 317 women not on vitamin D but on calcium is 26%, then there is 80% power to detect a 2575 mean percent density in the 317 women on vitamin D and on calcium of 15% (vitamin D effect 2576 in the presence of calcium is 26 – 3 – 8 = 15%). 2577 2578 14.3 Kidney Function with Vitamin D/Calcium 2579 This sub-study will investigate the effect of vitamin D and calcium supplementation on measures 2580 of renal function and blood pressure. 2581 2582 Subjects in the parent study who consent to this ancillary study will undergo the following 2583 additional procedures at their end of treatment (EOT) visit (either Year 3 or Year 5 EOT): 2584 • Urine collection for measuring microalbuminuria 2585 • Blood pressure measurement 2586 • An additional vial of blood will be collected for measuring markers of kidney 2587 function (see below) 2588 2589 In addition, creatinine and calcium measurements previously performed on all randomized 2590 subjects in the parent study at baseline, year 1, and year 3 (for subjects with a 5 year follow-up 2591 colonoscopy) will be used in these analyses. Creatinine and calcium measurements will also be 2592 performed on blood samples collected on all randomized subjects in the parent study at the Year 2593 3 and Year 5 End-of-Treatment visits and used in these analyses. 2594 2595 14.3.1 Background 2596 Chronic kidney disease (CKD), assessed by either the presence of microalbuminuria or 2597 decreased kidney function, affects over 23 million people in the United States and is associated 2598 with a high risk of hypertension, cardiovascular events, mortality and progression to end-stage 2599 renal disease. Known risk factors for kidney disease include the presence of high blood pressure, 2600 diabetes mellitus, dyslipidemia, non-white race and older age. Novel risk factors for kidney 2601 disease progression are continuously being identified, including low 25-hydroxyvitamin D 2602 (25(OH)D) levels and elevated serum phosphate and FGF-23 levels.175-177 2603 Animal studies suggest plausible mechanisms whereby low 25(OH)D levels and high 2604 phosphate levels may be harmful. Activated vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), 2605 suppresses renin biosynthesis in mice and vitamin D deficiency stimulates renin production.178 In 2606 different animal models, 1,25(OH)2D or its analogs reduced proteinuria levels, preserved

v 20.0 9-11-14 57

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2607 glomerular podocyte structure, 179 decreased levels of transforming growth factor β1, an inducer 2608 of renal fibrosis, and inhibited mesangial cell proliferation, a marker of renal injury. 180 High 2609 phosphate levels in rats lead to worse kidney function.181 In the setting of kidney disease, 2610 vitamin D supplementation may lead to higher phosphate levels through increased gut absorption 2611 but calcium therapy can act as a phosphate binder in the gut and may prevent a rise in phosphate 2612 levels in those treated with vitamin D. 2613 High blood pressure can be both a consequence of kidney disease and can independently 2614 cause kidney disease. Low vitamin D levels have been associated with the development of 2615 incident high blood pressure. 182 Some trials of vitamin D therapy have noted lower blood 2616 pressures in the vitamin D treated group. 183-185 However, these trials had small sample sizes and 2617 were not powered to evaluate changes in blood pressure. 2618 Thus far, vitamin D and phosphate binding therapies have not yet been tested in a large 2619 randomized clinical trial powered to evaluate kidney disease outcomes. The Vitamin D/ Calcium 2620 Polyp Prevention Study is the ideal setting in which to test the effects of vitamin D and/or 2621 calcium on kidney function in a double blind, placebo controlled setting. 2622 2623 14.3.2 Objectives 2624 The primary aim is to investigate the effect of calcium and/or vitamin D on the prevalence of 2625 microalbuminuria, a marker of kidney damage, at the end of 3 or 5 years of treatment with 2626 calcium and vitamin D, calcium alone or vitamin D alone versus placebo. 2627 Hypothesis: Vitamin D treatment will be associated with a lower prevalence of 2628 microalbuminuria. 2629 2630 The secondary aims are as follows: 2631 1) To evaluate the effects of calcium and/or vitamin D on blood pressure at the end of 3 or 5 2632 years of treatment with calcium and vitamin D, calcium carbonate alone or vitamin D alone 2633 versus placebo. 2634 Hypothesis: Vitamin D therapy will be associated with significantly lower blood pressure. 2635 2) To evaluate the effects of calcium and/or vitamin D on kidney function as measured by 2636 serum creatinine measurements at baseline, year 1, year 3 and year 5 in all participants of the 2637 parent study with available creatinine measurements. Additional urine and serum markers of 2638 kidney function to be measured at the end of 3 to 5 years of treatment will include: 2639 a. Urine: markers of tubular damage including KIM-1 and NGAL 2640 b. Serum: creatinine (to be assayed at the lab used for baseline measurement),calcium 2641 and cystatin C, an additional marker of kidney function 2642 c. Serum: bone markers and other markers of kidney disease progression including 2643 serum phosphate, alkaline phosphatase, intact parathyroid hormone (PTH) and 2644 fibroblast growth factor-23 (FGF-23) levels. 2645 Hypothesis: Treatment with vitamin D and calcium will result in better kidney function 2646 compared to placebo. 2647 2648 14.3.3 Recruitment and Enrollment Procedures 2649 The parent study defines the pool of potential subjects and those subjects who are willing 2650 to participate will be consented at the in-person End-of-Treatment visit (which is part of the 2651 parent study procedures) prior to their 3-year or 5-year follow-up colonoscopy. Participants will 2652 be reimbursed $15 for their time and effort after completion of the specimen collection. 2653 2654 14.3.4 Specimen/data Collection and Analysis

v 20.0 9-11-14 58

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2655 Urine collection 2656 Subjects will be instructed on clean catch urine collection techniques and will be 2657 provided with a pre-labeled sterile container for urine collection. After they have completed the 2658 urine collection they will give the urine to the research coordinator for processing. 2659 2660 Blood pressure measurement 2661 An automatic Sphygmomanometer will be used to provide a uniform measurement of 2662 blood pressures. All staff will be trained in the proper procedure for blood pressure 2663 measurement. 2664 2665 Blood collection 2666 Additional blood samples will be collected for this sub-study while the participant is 2667 undergoing the Year 3 or Year 5 End-of-Treatment blood collection. 2668 2669 Processing and Shipping 2670 Specimens (aliquots of blood and urine) will be shipped from the Study Centers to the 2671 Clinical & Molecular Pharmacology Shared Resource Laboratory at the Dartmouth Coordinating 2672 Center. These specimens will then be batched and shipped to the Albert Einstein College of 2673 Medicine for analysis. 2674 Urines - Urine samples will be promptly frozen at -20°C or below pending transfer to a - 2675 70°C freezer for storage until shipment to the Coordinating Center. 2676 Bloods - Blood samples will be sent to local labs for analysis (creatinine) or promptly 2677 frozen at -20°C or below pending transfer to a -70°C freezer for storage until shipment to the 2678 Coordinating Center. 2679 2680 Analyses 2681 Measurement of Serum Creatinine, Calcium, Cystatin C, Phosphate. Alkaline phosphatase, 2682 FGF-23 and intact PTH 2683 At the Albert Einstein College of Medicine Institute for Clinical and Translational 2684 Research laboratory, specimens will be received and appropriately stored until analysis. 2685 Analyses will be batched and analyses will be performed once all of the specimens are received. 2686 Measurement of urinary albumin, creatinine, KIM-1 and NGAL 2687 Analyses will be batched and performed by the Albert Einstein College of Medicine 2688 Institute for Clinical and Translational Research laboratory using standard methods. 2689 2690 14.3.5 Sample Size and Power 2691 Of the 2259 participants in the parent study, we plan to recruit approximately 900 into 2692 this sub-study. In the Framingham Offspring cohort with a mean age of 59 years, 12.2% had 2693 microalbuminuria, defined as at least 30 mg/g creatinine. 186 In the NHANES III survey, which 2694 includes younger participants, 8.5% of participants had a urinary albumin:creatinine ratio greater 2695 than 30 mg/g.187 Assuming the background prevalence of microalbuminuria in the trial is 12% 2696 and an alpha of 0.05, we will have 80% power to detect a relative risk of 0.64 for 2697 microalbuminuria comparing the vitamin D groups to the placebo group with 900 participants. 2698 We will therefore aim to recruit 900 participants for the urinary albumin measurement. In the 2699 Framingham Offspring Study, the standard deviation for systolic blood pressure is approximately 2700 20 mm Hg. 186 Assuming an alpha of 0.05 and 900 participants, we will have greater than 85% 2701 power to detect a difference of 3 mm Hg between the vitamin D and placebo groups. For the

v 20.0 9-11-14 59

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2702 repeated measures analysis for creatinine we will only use data from participants who have had 2703 all of their creatinines measured at the same laboratory (n≈1500). Having a sample size of 1500 2704 for repeated measures analysis to evaluate changes in creatinine with an alpha of 0.05 gives us 2705 90% power to detect a difference of 0.1 mg/dL in creatinine measurements. Evaluating changes 2706 in bone serum biomarkers, assuming an alpha of 0.05, we have >90% power to detect a 2707 difference of 1 standard deviation between the groups. 2708 2709 14.3.6 Statistical Analyses 2710 Specific Aim #1: All analyses will be performed and outliers will be verified by re-assay, if 2711 urine is available. Because this is a post-hoc analysis of a randomized clinical trial and data will 2712 not be available on the entire clinical trial population, we will create a table to evaluate whether 2713 baseline characteristics are equally distributed among the participants in the sub-study. The 2714 proportion of participants in each randomized group with microalbuminuria (defined as >30 2715 mg/g creatinine) will be compared using a chi-square test. If there are imbalances in the baseline 2716 characteristics, we will perform a logistic regression with the outcome albuminuria adjusting for 2717 characteristics that were imbalanced at baseline. In addition, we will compare medication use at 2718 follow-up to evaluate whether there were imbalances in NSAID and ACE inhibitor and 2719 Angiotensin Receptor Blocker (ARBs) use between the randomized groups. 2720 2721 Specific aim #2: The mean blood pressure of the 3 measurements will be used for all analyses. 2722 Baseline characteristics of the randomized groups included in this sub-study will be compared. 2723 In addition, the use of anti-hypertensive medications at the time of the blood pressure 2724 measurement will be compared between the groups. We will first compare the systolic and 2725 diastolic blood pressures among the 4 randomized groups using ANOVA. We will also compare 2726 vitamin D to no vitamin D using Student’s t-test. If characteristics are not balanced between the 2727 groups, we will use multivariable adjusted linear regression to evaluate whether treatment had an 2728 effect on systolic and diastolic blood pressures separately. 2729 2730 Specific aim #3: All data will be checked for outliers and specimens will be re-assayed for 2731 extreme outliers. Creatinine measurements will be converted to estimated glomerular filtration 2732 rates using the CKD-Epi equation. 188 We will compare the prevalence of CKD (defined as an 2733 estimated GFR <60 ml/min/1.73m2) between the 4 treatment arms using chi-square test. We will 2734 also used random effects models with repeated creatinine measures which can account for 2735 missing values to compare serial creatinines between the 4 treatment groups. To evaluate 2736 whether calcium and/or vitamin D supplementation over 5 years affected serum phosphate, 2737 alkaline phosphatase, intact PTH and FGF-23 levels, we will compare levels between the 2738 intervention groups and the placebo group using appropriate methods for skewed data. 2739 2740 14.3.7 Human Subjects Research 2741 Human Subjects Involvement and Characteristics 2742 The participants in this ancillary study are already enrolled in a 3 to 5-year randomized 2743 clinical trial of calcium and/or vitamin D. They have already consented to participate in the 2744 parent study. For this ancillary study, they will undergo an additional informed consent 2745 procedure during their end-of-treatment visit for the parent study and, if consented, will have 2746 their blood pressure measured, give a urine sample and have additional blood samples taken at 2747 the end-of-treatment blood draw for the parent study.

v 20.0 9-11-14 60

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2748 We expect that the population for this sub-study will be similar to that of the overall 2749 parent study. The mean age at enrollment was 58.1 (standard deviation 6.8), the participants 2750 were 37% female and 83% white, 8.1% black, 8.5% other race and 6.5% of Hispanic ethnicity. 2751 The study will take place at the sites of the parent trial: Dartmouth Hitchcock Medical 2752 Center, Cleveland Clinic Foundation, University of Colorado, Emory University, University of 2753 Iowa, University of Minnesota, University of North Carolina, University of South Carolina, 2754 University of Southern California, University of Texas and University of Puerto Rico and their 2755 related practice sites. 2756 2757 Sources of Materials 2758 The participants in this sub-study will give urine and blood samples and will have their 2759 blood pressures measured. The parent study is collecting information on demographics, co- 2760 morbid health conditions, medications and other health information. The new data will be linked 2761 to existing data via unique identifiers. 2762 2763 Potential Risks 2764 The additional physical risks associated with the sub-study are those associated with the 2765 collection of the urine, blood and blood pressure, which are outside of the standard of care. 2766 Drawing blood, or venipuncture, may result in bruising to the arm or on rare occasions, 2767 vasovagal syncope. Blood pressure measurements involve some discomfort as the 2768 sphygmomanometer is inflated. There are no significant risks to a spot urine collection. All these 2769 risks are detailed in the informed consent document. There may be additional risks of breach of 2770 confidentiality with the additional measurements taken. All study personnel are already trained 2771 in the importance of maintaining confidentiality and all data for analysis at the Albert Einstein 2772 College of Medicine will be de-identified. 2773 2774 Adequacy of the Protection against Risks 2775 Recruitment and Informed Consent - All patients included in this sub-study have already 2776 undergone 3 or 5 years of a randomized clinical trial with blood testing and colonoscopies. They 2777 will undergo a separate informed consent process for this sub-study detailing the rationale for the 2778 study and risks and benefits associated with participation. 2779 2780 2781 Protection against Risk - All staff are trained to maintain complete confidentiality. 2782 All data transferred to the Albert Einstein College of Medicine will be completed de- 2783 identified. Venipuncture and blood pressure measurements will be conducted by 2784 experienced phlebotomist and technician with the patient seated. The Data and Safety 2785 Monitoring Committee for the parent trial will serve in this capacity for the ancillary 2786 study, by monitoring for potential complications and recommending whether the trial 2787 should continue. 2788 2789 Potential Benefits of the Proposed Research to the Subject and Others - The participants of 2790 this trial will likely not directly benefit from the kidney functions ancillary study. However, the 2791 results of this trial may impact the treatment for patients with kidney disease and high blood 2792 pressure with an inexpensive therapy, calcium and vitamin D. 2793 2794 Importance of Knowledge to be Gained (Risk: Benefit Ratio) - High blood pressure, kidney 2795 disease and microalbuminuria are all associated with high risk of cardiovascular disease and

v 20.0 9-11-14 61

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2796 mortality. A new, inexpensive therapy to treat high blood pressure, prevent the progression of 2797 kidney disease and/or albuminuria would have far-reaching public health implications. The risks 2798 to the participant for this ancillary study are fairly minimal and involve the collection of blood, 2799 urine and blood pressure measurements at a single time point at the end of a randomized clinical 2800 trial evaluating colonic polyp recurrence. The risks detailed above are fairly minimal in 2801 comparison to the knowledge to be gained. 2802 2803 Data Safety Monitoring Plan - The Data and Safety Monitoring Committee for the parent 2804 trial will serve in this capacity for the ancillary study, by monitoring for potential complications 2805 and recommending whether the trial should continue. 2806 2807 2808 Inclusion of Women and Minorities - We expect that the participants in the kidney 2809 function sub-study will have the same gender and race/ethnicity make-up as the parent 2810 trial. Therefore we expect 37% female participants and 83% white, 8.1% black, 8.5% 2811 other race and 6.5% of Hispanic ethnicity. We will not exclude any participants based on 2812 gender or race/ethnicity. 2813 2814 Inclusion of Children - Children are not enrolled in the parent study, the Vitamin D Polyp 2815 Prevention Trial because children generally do not get colonic polyps and if they do occur in 2816 children, they are part of a genetic syndrome and probably would not be affected by vitamin D 2817 and calcium supplementation. Therefore, no children will be recruited into the kidney functions 2818 sub-study. 2819 2820 14.4 Participant Beliefs and Bias Study 2821 This ancillary analysis is under the direction of Dr. Judith R. Rees at the Geisel School of 2822 Medicine at Dartmouth. 2823 2824 14.4.1 Background and rationale 2825 The randomized, placebo-controlled trial uses allocation concealment and blinding of 2826 participants and investigators to minimize bias in patient selection, adherence, and ascertainment 2827 of outcomes. Beliefs about the assigned treatment (allocation belief) and its efficacy (efficacy 2828 belief), and treatment preference may affect adherence, health behaviors, attrition, and health 2829 endpoints. We will study the relations between these beliefs, blinding, adherence and health 2830 outcomes. Our findings will help develop strategies to prevent and/or correct for belief-related 2831 biases in future trials. 2832 2833 14.4.2 Objectives 2834 Using data already collected during the trial (Section 5.15), we will: 2835 1. Identify symptoms and other predictors of subsequent allocation belief at randomization, year 2836 2, and end of treatment (EOT). 2837 2. Assess how subjective and objective health outcomes are affected by allocation beliefs, 2838 efficacy beliefs, and preference. 2839 3. Assess the effects of allocation beliefs, efficacy beliefs, and preference on adherence. 2840 2841 14.4.3 Data collection

v 20.0 9-11-14 62

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2842 Data collection (approved previously) is conducted as part of the routine interviews for the 2843 parent study, as well as separate mail-back study methods surveys. 2844 2845 14.4.4 Analyses 2846 Allocation beliefs will be classified as “Believe Active” (A) for any active treatment option in 2847 the 4-arm study or calcium+vitamin D in the 2-arm study;. “Believe Placebo” (P) for placebo in 2848 the 4-arm study; calcium in the 2-arm study; or “Don’t Know (DK). We will analyze all subjects, 2849 and separately those in the 4-arm and 2-arm studies. We will assess the distribution of forced 2850 guesses of those who said DK and incorporate the appropriate analyses and sensitivity analyses 2851 to show the effect of pooling forced guesses with Active or Placebo guesses. A 2-sided p-value 2852 <0.05 will be considered statistically significant. We will not adjust for multiple testing. Our 2853 analyses will be based primarily on describing the data and regression modeling. . 2854 2855 Aim 1. Identify predictors of allocation belief at randomization, year 2, and EOT. 2856 At different times during the trial, we will classify the reasons subjects provided for their 2857 allocation beliefs and explore the factors that predict beliefs. We will explore the factors 2858 associated with misunderstanding of equipoise and randomization. 2859 2860 Hypothesis 1. Allocation beliefs change during the course of a double-blinded trial. We will 2861 describe the distributions of beliefs, changes in beliefs from randomization to year 2 [EOT], and 2862 use regression models to identify factors related to changes of belief. 2863 2864 Aim 2. Examine how subjective and objective health outcomes are affected by allocation beliefs, 2865 efficacy beliefs, and preference. We will assess how beliefs affect subsequent symptoms and on 2866 colorectal adenoma, using multivariable regression. 2867 2868 Hypothesis 2a. Allocation beliefs at randomization [year 2, EOT] predict the occurrence of each 2869 of five specified health effects, after adjustment for a history of that same health effect. We will 2870 use multivariable regression models of the relations between beliefs and subsequent symptoms 2871 identified during routine bi-annual telephone surveys. 2872 2873 Hypothesis 2b. Allocation beliefs at randomization [year 2, EOT] predict a significant change in 2874 each of five SF-36 scores during the study, independently, or by interaction with efficacy beliefs 2875 about that health effect, after adjusting for baseline score and randomized treatment. Hypothesis 2876 3 is analogous to Hypothesis 2, but uses SF-36 data on general health, physical functioning, 2877 bodily pain, vitality, and mental health. 2878 2879 Aim 3. Examine how adherence is affected by allocation/efficacy beliefs, and preference. 2880 We will model subjects’ adherence, in relation to their preferences, efficacy beliefs, and 2881 allocation beliefs at different times, and demographic and medical factors. 2882 2883 Hypothesis 3. The proportion of pills taken during run-in is associated with allocation beliefs at 2884 randomization, after adjustment for baseline efficacy beliefs and preferences. We will develop a 2885 model for adherence including allocation belief; efficacy belief, preference, and repeat these 2886 analyses for adherence averaged over the first two years. We will develop models to predict 2887 completion of the trial on study pills. 2888

v 20.0 9-11-14 63

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2889 14.4.4 Sample size 2890 We will use all 2,813 enrollees where possible, and for other analyses, the 2,259 randomized 2891 subjects. For continuous outcome measures, we will have 90% power to detect a difference of 2892 0.09 SD at baseline, and 0.11 SD at year 2, based on t-tests comparing allocation beliefs. For 2893 multivariable analyses, assuming that each additional predictor variables requires a 10% increase 2894 in sample size, we will be able to detect adjusted differences of 0.15 SD at baseline and 0.20 SD 2895 at Year 2 or EOT in models containing 20 additional predictors. For binary outcomes, at 2896 baseline, we will have 90% power to detect an odds ratio <1.3 at baseline, year 2 and EOT. For 2897 multivariable analyses assuming the outcome has prevalence of just 10% of the total sample, 2898 model estimates will be reliable with 28 predictors for baseline data models and 16 predictors for 2899 year 2 models. Hence our study sample is sufficient for the analyses described. 2900 2901 14.4.5 Additional Risks 2902 This sub-study presents no additional physical risk to the subjects. Confidentiality of data is well 2903 protected, as described earlier. 2904 2905 14.4.6 Benefits and importance of knowledge to be gained 2906 There is no direct benefit to subjects. Our results may improve randomized controlled trial 2907 methodology; in that case, society will benefit from improved trials in the future. 2908 2909 14.4.7 Inclusion of women, minorities 2910 The sub-study uses data from all subjects in the parent study. 2911 2912 2913 14.5 Colds and Flu study 2914 This ancillary study is under the direction of Dr. John A. Baron, PI for the parent study at the 2915 Geisel School of Medicine at Dartmouth. 2916 2917 14.5.1 Background and rationale 2918 Observational studies indicate that vitamin D may play a role in the prevention of infectious 2919 diseases. We will evaluate the effects of vitamin D supplementation on the incidence and 2920 duration of upper respiratory tract infection (URTI), influenza-like illness (ILI), and acute 2921 gastrointestinal illness (AGI), by adding surveillance for these three illnesses to our on-going 2922 randomized controlled clinical trial of vitamin D and/or calcium supplementation for the 2923 prevention of colorectal adenomas. 2924 2925 14.5.2 Objectives 2926 Our specific aims are: 2927 To collect health dairies recording the incidence and duration of common infectious illnesses 2928 in approximately 800 subjects enrolled in our clinical trial of vitamin D and calcium. 2929 To assess the effects of vitamin D supplementation on the risk of upper respiratory tract 2930 infections and explore effects on gastrointestinal illnesses, using formal standardized case 2931 definitions of URTI, ILI, and AGI 2932 2933 Our primary hypotheses are: 2934 Supplementation with vitamin D3 (1000 IU/day) reduces the number of winter episodes of 2935 URTI.

v 20.0 9-11-14 64

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2936 Supplementation with vitamin D3 (1000 IU/day) reduces the number of winter days affected 2937 by URTI. 2938 2939 Our exploratory hypotheses are: 2940 Supplementation with vitamin D3 (1000 IU/day) reduces the number of episodes of URTI 2941 over a year of observation. 2942 Supplementation with vitamin D3 (1000 IU/day) reduces both the number of winter episodes 2943 of ILI and the number of winter days affected by that illness. 2944 Supplementation with vitamin D3 (1000 IU/day) reduces both the number of episodes of AGI 2945 and the days affected by that illness. 2946 2947 In addition, we will (i) compare the recent reporting of colds and ILI by subjects telephoned 2948 every 6 months among those randomized to vitamin D3 (1000 IU/day) supplementation or 2949 placebo; and (ii) examine the impact of serum levels of 25-hydroxyvitamin D3 (25-OHD) on 2950 subject’s experience of symptoms during the study. 2951 2952 14.5.3 Data collection 2953 Questionnaires (implied consent) were administered as described below. 2954 2955 Colds and Flu Questionnaires 2956 Subjects will be asked to complete up to eighteen monthly questionnaires distributed from the 2957 Dartmouth Project Coordination Center to collect information related to colds, flu and flu-like 2958 illnesses. 2959 2960 Dartmouth Project Coordination Center staff will communicate directly with subjects via phone 2961 or e-mail for reminders or clarifications about questionnaires. Local study coordinators may be 2962 asked to assist in calling or e-mailing to remind subjects who have not returned questionnaires or 2963 have been ambiguous about participation, and may discuss the subject’s Colds and Flu 2964 Questionnaire participation at their regular study Interval phone calls. 2965 2966 The Introductory mailing will include: 2967 • COLDS AND FLU QUESTIONNAIRES (Part 1: Daily Health Diary; Part 2: 2968 Questionnaire) with prepaid return envelopes 2969 • Invitation Letter 2970 • Information Sheet 2971 • Opt-Out Postcard 2972 • Participant Instructions 2973 • Daily Health Diary Example 2974 • Study logo refrigerator magnet 2975 • $5.00 (in cash or gift card) for reviewing materials and considering participation 2976 2977 The subject may choose to complete the questionnaire(s) (implied consent) or may opt-out via 2978 postcard. Alternately, per local procedures, the subject may be consented in person, by mail or 2979 by phone to participate in the new questionnaires. Data will be collected regarding daily cold 2980 and flu symptoms, vaccinations, relevant diagnoses, medications prescribed or supplements 2981 taken for symptoms reported, and information about travel. The paper questionnaires will be 2982 returned by mail (using the prepaid envelopes provided) to the Dartmouth Project Coordination

v 20.0 9-11-14 65

Downloaded From: https://jamanetwork.com/ on 09/26/2021 2983 Center for data entry. Alternately, the subject may choose to complete the questionnaires by 2984 direct web entry. 2985 2986 Subsequent mailings. 2987 Upon receipt of questionnaire(s) from the introductory mailing, subsequent mailings of up to 2988 three questionnaires will be mailed to the subject (or will be available on-line) until up to 2989 eighteen questionnaires have been distributed. The subsequent mailings will include: 2990 • Subsequent mailing cover letter 2991 • COLDS AND FLU QUESTIONNAIRE(S) with prepaid return envelope(s) 2992 • $5.00 (in cash or gift card) to reimburse subject for each completed questionnaire returned at 2993 the time of the subsequent mailing 2994 • Instructions for completing web-based questionnaires 2995 2996 Subjects who did not participate when only paper questionnaires were available will be given the 2997 opportunity to participate in the on-line version by opting in via postcard, phone call or e-mail to 2998 the Dartmouth Project Coordination Center. 2999 3000 Internet option mailing. 3001 A mailing will be sent to announce the availability of the web-based Questionnaire to all subjects 3002 who did not participate when only the paper questionnaire was available. This mailing will 3003 include: 3004 • Cover letter 3005 • Opt-In Postcard for subjects who did not participate when only the paper questionnaire was 3006 available 3007 • Instructions for completing web-based questionnaires 3008 3009 Reminders - A reminder letter, e-mail or phone call will be generated from the Dartmouth 3010 Project Coordination Center or local study coordinator if an expected paper questionnaire is not 3011 returned or an online questionnaire not submitted. 3012 3013 Final mailing - We will continue to collect up to five additional questionnaires (not to exceed a 3014 total of eighteen) after the completion of the treatment phase because of the long half-life of 3015 vitamin D. Upon receipt of the final questionnaires, a letter will be sent with any remaining gift 3016 cards for questionnaires completed and submitted. 3017 3018 25-OHD was measured as described in Sections 5.12 and 9.1 - 9.3. 3019 3020 14.5.4 Analyses 3021 URTI: We define a common cold or upper respiratory tract infection (URTI) as the presence of 3022 2 or more of the following: runny nose, nasal congestion, sneezing, sore throat, cough, swollen 3023 or tender neck glands. 3024 ILI: We define Influenza-like illness (ILI) as fever, plus 2 or more of the following: sore throat, 3025 cough, muscle ache, headache. 3026 AGI: We will base our definition of acute gastrointestinal illness on Payment’s “highly credible 3027 gastrointestinal illness”: either 1) vomiting or watery diarrhea; or 2) nausea or soft diarrhea with 3028 abdominal cramps. 3029

v 20.0 9-11-14 66

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3030 Further, our definition of an episode of illness requires 6 symptom-free days before a subsequent 3031 episode will be said to have occurred. We will require this symptom-free interval because we 3032 assume that symptoms occurring toward the end of an illness may not necessarily fulfill the case 3033 definition but will still be part of the episode. For example, on days 1, 2 and 3, a participant’s 3034 symptoms fulfill the definition of a common cold (sore throat and nasal congestion). On days 4 3035 and 5 only a mild sore throat persisted, but the nasal congestion recurred on days 6 and 7, which 3036 together with the sore throat fulfill the case definition for those two days. According to our 3037 definitions, this constitutes only one episode of URTI. For the purposes of estimating “days of 3038 illness”, we will use our case definitions strictly, so that day 4 and 5 are excluded, and there were 3039 only 5 days of URTI altogether. 3040 3041 Statistical methods 3042 Primary end points will be the number of episodes of each type of illness and the total number of 3043 days of affected by the illness. Because of the seasonally low vitamin D levels in winter, our 3044 main hypothesis is that supplementation will have an effect during those months, rather than 3045 during the summer. 3046 3047 For the analysis of the number of illness episodes, we will use Poisson regression to estimate the 3048 effect of treatment assignment to vitamin D. The primary analyses will be by intention-to-treat – 3049 i.e. we will ignore subject compliance in the analysis, which will be based solely on the 3050 randomized assignment. We adjust for calcium treatment assignment in all analyses. We will 3051 also pay special attention to over-dispersion or under-dispersion, as assessed by the usual chi- 3052 squared testing. If this is statistically significant, we will use robust methods to estimate standard 3053 errors and recalculate the confidence interval for the treatment effect. Other analyses e.g. of the 3054 days affected by illness will proceed in the same spirit. 3055 3056 We will compare randomized treatment groups in relation to the probability of any self reported 3057 health outcome ascertained during 6 monthly interviews (colds, ILI). 3058 3059 14.5.5 Sample size 3060 Based on national data and data from our own study, we estimate that placebo subjects 3061 experience between 0.6 and 0.9 URTI winter UTRI per year on average. With our expected 3062 recruitment, we will have excellent statistical power to detect vitamin D relative risks of 0.7 or 3063 lower, and adequate statistical power for relative risks of 0.8. Based on national data, we 3064 estimate that placebo subjects experience between 1 and 3 days affected by a winter URTI per 3065 year on average. With our expected recruitment, we will have excellent statistical power to 3066 detect vitamin D relative risks of 0.8 or lower. 3067 3068 14.5.6 Additional Risks 3069 This sub-study presents no additional physical risk to the subjects. Serum 25-OHD was already 3070 collected for the parent study. Confidentiality of data is the primary concern, but the data are not 3071 sensitive and are well protected as described earlier. 3072 3073 14.5.7 Benefits and importance of knowledge to be gained 3074 There is no direct benefit to subjects. We aim to describe whether vitamin D prevents colds and 3075 flu; the answer to this question will benefit society as a whole. 3076 3077 14.5.8 Inclusion of women, minorities

v 20.0 9-11-14 67

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3078 The sub-study was available to subjects of all demographic categories within the parent study. 3079 3080 14.6 The Dartmouth Polyp Prevention Biospecimen Repository 3081 All subjects will be invited to contribute blood samples, and extracted DNA, as well as 3082 colorectal tissue specimens in the parent study, and biospecimens collected in added ancillary 3083 studies, for future research on colorectal neoplasia or other disorders common in the study 3084 population. Their decision will be documented as part of the informed consent. 3085 3086 In the parent study, for subjects who agree to future research on their blood samples, 3087 approximately 12 additional 1.0 or 0.5 ml aliquots of plasma or serum will be obtained at each 3088 blood draw and shipped to the biospecimen storage facility at the NCCC Clinical & Molecular 3089 Pharmacology Shared Resource Laboratory. In addition, DNA that is extracted from these 3090 subject’s blood samples, and that is not used up during the VDR genotyping, will be stored for 3091 future research. 3092 3093 In the parent study, for subjects who agree to future research on the tissue samples collected 3094 during routine clinical care, tissue blocks will be requested from the appropriate pathology 3095 laboratories and shipped to the biospecimen storage facility at the NCCC Clinical & Molecular 3096 Pharmacology Shared Resource Laboratory. If the pathology laboratory requests return of the 3097 tissue blocks, they will be used to obtain sections or cores for slides or Tissue Micro Arrays. 3098 Tissue blocks, sections and cores will be stored at the NCCC Clinical & Molecular 3099 Pharmacology Shared Resource Laboratory for future research. 3100 3101 In ancillary studies, unless otherwise provided in the protocol and consents, all biospecimens 3102 collected will be shipped to the biospecimen storage facility at the NCCC Clinical & Molecular 3103 Pharmacy prior to shipment to collaborating labs. Any biospecimen that is not used up in lab 3104 analyses will be stored for future research at the NCCC Clinical & Molecular Pharmacology 3105 Shared Resource Laboratory. 3106 3107 All future research utilizing these biospecimens will require IRB review and will be consistent 3108 with the intent of the informed consent obtained from the subject. 3109 3110 14.7 Genetic variant influencing response to vitamin D and calcium 3111 Polymorphisms in the vitamin D receptor (VDR) will be genotyped as part of the primary 3112 protocol for all randomized subjects. In this ancillary aim, we will investigate the association of 3113 common genetic variation in other genes involved in calcium or vitamin D metabolism or 3114 signaling with the response to vitamin D supplementation (increase in serum 25-hyrdoxy- 3115 vitamin D) and the occurrence of colorectal adenomas. Genes to be investigated are likely to 3116 include, but are not limited to, the vitamin D binding protein (DBP), retinoid X receptor (RXR), 3117 vitamin D 25-α-hydroxylase (CYP2R1), vitamin D 1-α-hydroxylase (CYP27B1), vitamin D 24- 3118 α-hydroxylase (CYP24A1), 7-dehydrocholesterol reductase (DHCR7), the calcium-sensing 3119 receptor (CaSR), the apical sodium-dependent bile acid transporter (ASBT) and the apical 3120 calcium channel (TRPV6). There is no separate consent for this ancillary aim. Instead, only 3121 subjects that authorized storage of their blood specimens for future research, including genetic 3122 analyses, will be included in this analysis. Notably, these metabolic genes are thought to be low 3123 penetrance with only modest effects on colorectal carcinogenesis. Confidentiality is protected in 3124 the following ways: 1- DNA samples will be de-identified and labeled only with unique barcode

v 20.0 9-11-14 68

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3125 numbers, 2- a de-identified “Limited Data Set”, as defined by HIPAA, will be used to examine 3126 the results of these genetic analyses. In addition, as described in section 14.7 below, we have a 3127 obtained a Certificate of Confidentiality for this study, as issued by the National Institutes of 3128 Health, that protects subjects from the release of any potentially sensitive genetic information to 3129 persons not associated with this research. 3130 3131 14.8 Early detection of colorectal neoplasia with circulating tumor DNA (ctDNA) 3132 The main objective of these analyses is to develop clinical tests that can be used to identify the 3133 presence of colorectal neoplasms at a stage when the tumors can be cured by surgery. State-of- 3134 the-art technologies will be used to test for the presence of mutant DNA template molecules in 3135 plasma specimens that have been coordinated with tissue specimens collected at the targeted 3136 colonoscopy. Paired samples of tissue and plasma collected prior to the development of 3137 clinically significant colorectal tumors will allow analyses of a critical aspect of the entire 3138 approach: that circulating tumor DNA can be detected prior to any symptoms, well before they 3139 were discovered through conventional methods. The criteria for choosing paired specimens will 3140 be based on the size of the colonoscopy lesion, the pathology diagnosis, and the timing of study 3141 blood collected prior to the colonoscopy at which the lesion of interest or cancer was removed, 3142 together with subject consent for future research on blood and tissue. Targeted tissue blocks 3143 obtained from local pathology labs will be shipped from local study centers to the Dartmouth 3144 coordination center for administrative processing before shipping to the lab performing the 3145 analyses. Concurrently, targeted plasma specimens stored at the NCCC Clinical & Molecular 3146 Pharmacology Shared Resource Laboratory will be processed for shipping to the lab. All tissue 3147 specimens will be returned to the Dartmouth coordination center for administrative processing 3148 before shipping back to the local study center/pathology lab, if requested. Blood samples will 3149 likely be exhausted in the analyses, but any remaining samples will be returned to the NCCC 3150 Clinical & Molecular Pharmacology Shared Resource Laboratory. 3151 3152 14.9 Metabolomic Study of the Effects of Vitamin D and Calcium Supplementation 3153 The objective of this ancillary study is to use a global metabolomics approach to better 3154 understand the independent and synergistic antineoplastic actions and other effects of vitamin D 3155 and calcium in humans. The aims are to elucidate 1) the individual and combined effects of 3156 vitamin D and calcium on metabolomic pathways in humans (thus providing a more 3157 comprehensive assessment of the benefits and risks of these heavily promoted agents as 3158 supplements), 2) whether these metabolomic changes correlate with genetic variation and 3159 systemic and tissue-specific biomarkers in humans (and thus their direct relevance to the 3160 chemoprevention of colorectal neoplasms), and 3) whether the metabolomic changes predict the 3161 occurrence of sporadic colorectal adenomas in humans, thus paving the way for the development 3162 of treatable biomarkers of risk for colorectal neoplasia that are analogous to lipid profiles for the 3163 prevention of ischemic heart disease. In this pilot study we will investigate the effects of 3164 supplementation with vitamin D and/or calcium on individual metabolites and metabolic 3165 pathways in a sub-set of participants (n = 120) who were enrolled in the Calcium/Vitamin D, 3166 Biomarkers, and Colon Polyp Prevention ancillary study (see section 14.4 below.) There is no 3167 separate consent for this ancillary study; only subjects that authorized the storage of their blood 3168 specimens for future research will be included in this analysis. The aims will be accomplished 3169 by conducting state-of-the-art, high-resolution metabolomic profiling using liquid 3170 chromatography-Fourier transform mass spectrometry (LC-FTMS) analysis of blood plasma 3171 collected before and after supplementation with vitamin D3 and/or calcium. For this purpose 3172 blood samples will be shipped from the Dartmouth Biorepository to the collaborating laboratory

v 20.0 9-11-14 69

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3173 at Emory University (Dr. Dean Jones), where the metabolomic analyses will be performed. 3174 3175 14.10 Vitamin D3, Calcium and Biomarkers of Inflammation and Gut Barrier Function 3176 The objective of this ancillary study is to test a novel hypothesis that calcium and vitamin D 3177 supplementation strengthens the gut mucosal barrier as indicated by modulation of cell cycle, 3178 cell adhesion, receptors responsive to bacterial components, and markers of intestinal and 3179 systemic inflammation and colonic permeability in humans. The aims of the proposed study are 3180 1) in biopsies of normal-appearing rectal mucosa, to obtain data on the separate and joint effects 3181 of calcium and vitamin D3 supplementation on biomarkers of gut barrier function (Ki-67/MIB-1, 3182 caspase-3, TLR4, TLR5, ZO-1, claudin-1, occludin, mucin-17, E-cadherin, and β-catenin) and 3183 inflammation (NFκB and TNFα); 2) in blood samples, to obtain data on the separate and joint 3184 effects of calcium and vitamin D supplementation on biomarkers of inflammation (GM-CSF, 3185 IFNγ, TNFα, IL-6, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p40, IL-12p70 and IL-17) and gut barrier 3186 function (specific IgG and IgA to lipopolysaccharide [LPS] and flagellin, LPS binding protein 3187 [LBP], and intestinal fatty acid binding protein [I-FABP]); 3) to investigate whether the findings 3188 for the tissue-specific biomarkers are correlated with circulating biomarkers of gut barrier 3189 function and inflammation; and 4) to obtain preliminary data on whether changes in the 3190 biomarker levels are associated with decreased sporadic colorectal adenoma recurrence. The 3191 proposed scope of work is limited to laboratory and statistical analyses using already collected 3192 biological samples and questionnaire data. In this adjunct study, the blood and tissue assays will 3193 be performed in a sub-set of participants (approximately 460) who were enrolled in the 3194 Calcium/Vitamin D, Biomarkers, and Colon Polyp Prevention ancillary study (see section 14.4 3195 below). There is no separate consent for this ancillary study; only subjects who authorized the 3196 storage of their blood and tissue specimens for future research will be included in this analysis. 3197 The aims will be accomplished by conducting immunoassays to measure biomarkers in tissue or 3198 blood plasma/serum samples collected before and after supplementation with vitamin D3 and/or 3199 calcium. The blood samples are stored at the biospecimen storage facility at the NCCC Clinical 3200 & Molecular Pharmacology Shared Resource Laboratory and tissue samples are stored at Dr. 3201 Bostick’s Molecular Epidemiology and Biomarkers Research Laboratory, Emory University. 3202 3203 14.11 Mechanisms of obesity-related colorectal carcinogenesis 3204 The objective of this ancillary study is to understand how obesity contributes to colorectal cancer 3205 risk. Specifically, using data and biospecimens from the Vitamin D/Calcium Polyp Prevention 3206 Study, we will investigate which biological pathways mediate the increased risk of colorectal 3207 neoplasia that is associated with obesity by assessing the following serum biomarkers: 1) 3208 inflammatory cytokines secreted by adipose tissue (interleukin-6, soluble transforming growth 3209 factor receptor-2), 2) metabolically active adipokines (leptin, adiponectin), 3) insulin resistance 3210 and related factors (homeostatic model assessment – insulin resistance, insulin-like growth factor 3211 1, insulin like growth factor binding protein-3), 4) vitamin D status (25-hydroxy vitamin D), and 3212 5) sex hormones (testosterone, estradiol, sex hormone binding globulin). The aims are to assess 3213 the individual associations of these biomarkers with risk of colorectal adenomas, to quantify the 3214 proportion of the BMI-colorectal adenoma association that can be attributed to each of the 3215 biomarkers and to delineate the downstream mediators of the dominant biomarkers. There is no 3216 separate consent for this ancillary study; only subjects who authorized the storage of their blood 3217 and tissue specimens for future research will be included in this analysis. Biomarker assays will 3218 be conducted on de-identified blood serum/plasma samples at collaborating laboratories and any 3219 unused sample will be returned to the Dartmouth Biorepository. 3220

v 20.0 9-11-14 70

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3221 14.12 Sex hormones and Colorectal Neoplasia 3222 The objective of this study is to understand how reproductive factors modulate risk of colorectal 3223 adenomas and serrated polyps in both women and men. Specifically, we will assess: 1- 3224 associations between the risk of colorectal adenomas and colorectal serrated polyps in women, 3225 and age of menarche, age of menopause, gravidity, parity, oral contraceptives use, and hormone 3226 replacement therapy use; 2- associations of circulating free testosterone index, free estradiol 3227 index, estrone, and dihydroepiandrosterone sulfate with risk of incident colorectal adenomas and 3228 serrated polyps separately in men and women; 3- associations of adenoma and serrated polyp 3229 occurrence with polymorphisms in the genes for the androgen receptor (AR) and estrogen 3230 receptor beta (ESR2), separately in men and women. This research will use data collected in our 3231 four completed adenoma prevention trials and biospecimens from the two most recent studies 3232 (Aspirin/Folate Polyp Prevention Study and Vitamin D/Calcium Poly Prevention Study). Blood 3233 samples will be used to measure sex hormone levels and DNA will be used to genotype AR and 3234 ESR2 polymorphisms. De-identified blood or DNA samples will be sent to collaborating 3235 laboratories for these analyses and any unused sample will be returned to the Dartmouth 3236 Biorepository. 3237 3238 14.13 Interaction of Vitamin D and Calcium with Magnesium and Colorectal Adenoma 3239 Recurrence 3240 In this project, we will evaluate if use of calcium and/or vitamin D supplementation reduces risk 3241 of colorectal adenoma recurrence in subgroups of adenoma patients. Specifically, we will 3242 examine: 1) whether vitamin D supplementation reduces risk of colorectal adenoma recurrence 3243 when baseline levels of serum magnesium or magnesium intake are high or calcium/magnesium 3244 intake ratio is low; 2) whether the effect of vitamin D supplementation on risk of colorectal 3245 adenoma recurrence differs by serum levels of vitamin D metabolites which are affected by 3246 magnesium (e.g. serum 24,25(OH)2D and 24,25(OH)2D/25(OH)D ratio, and 3247 1,25(OH)2D/25(OH)D ratio); 3) if calcium supplementation reduces risk of adenoma risk when 3248 baseline calcium/magnesium intake ratio is low; 4) if biomarker patterns (e.g. proteomics and 3249 immunohistochemical assay of specific biomarkers including TRPV5, TRPV6, TRPM6, TRPM7, 3250 VDR, NKCC2, ROMK, CASR, SLC7A2, and SLC8A1) in adenoma tissues collected at baseline 3251 differ by the calcium/magnesium ratio; 5) if biomarker patterns in adenoma tissues can be used 3252 to predict the responses from calcium and/or vitamin D supplementation. This research will use 3253 data and biospecimens collected in two of our adenoma prevention trials (Calcium Polyp 3254 Prevention Study and Vitamin D/Calcium Poly Prevention Study). De-identified blood samples 3255 will be sent to collaborating laboratories for these analyses and any unused sample will be 3256 returned to the Dartmouth Biorepository. 3257 3258 14.14 Identification of prognostic epigenetic markers in biopsy tissue 3259 The ultimate objective of this project is to develop a prognostic test that will allow early 3260 identification of those patients who are most likely to experience advancement in the process of 3261 colorectal carcinogenesis. Prior research suggests that methylation of tumor suppressor genes in 3262 polyps is a causal event and that the methylation of certain loci is found in polyps with higher 3263 likelihood of progression. Our specific aims are to identify a panel of methylated loci that 3264 distinguish polyps that progress to advanced histologic outcomes from more benign lesions; and, 3265 to identify modifiable lifestyle factors associated with the progressive polyp methylation 3266 signature. The project will focus on a subset of study participants retrospectively selected based 3267 on their clinical outcome and/or on pathology of the archived tissue specimen. We will utilize 3268 portions of biopsy tissue samples that were archived from past colonoscopy procedures from

v 20.0 9-11-14 71

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3269 participants in the two most recent trials (Aspirin/Folate Polyp Prevention Study and Vitamin 3270 D/Calcium Poly Prevention Study). Tissue blocks archived at local pathology labs will be 3271 shipped from local study centers to the Dartmouth coordination center for administrative 3272 processing before shipping to the lab performing the assay. Tissue blocks will not be exhausted 3273 and will be returned to the study centers after sectioning, if requested. In addition to polyps, we 3274 will examine the methylation status of normal colorectal mucosa using biopsies collected at year 3275 3 in the Aspirin/Folate Study. High-throughput assays will be performed to assess the epigenetic 3276 status of each biopsy. 3277 3278 14.15 Oncoarray Genotyping 3279 The overarching goals of this research are to thoroughly investigate and identify susceptibility 3280 loci for colorectal cancer, to characterize the biologic basis of inherited susceptibility, and to 3281 recognize how genetic variation may be quantified and modified by genetic and environmental 3282 risk factors. For this purpose, we will genotype DNA isolated from participants in the two most 3283 recent studies, (Aspirin/Folate Polyp Prevention Study and Vitamin D/Calcium Poly Prevention 3284 Study). Genotyping will use the Infinium OncoArray-500K Beadchip, or other similar DNA 3285 microarray. The OncoArray contains 500,000 SNPs with a genome-wide backbone of 250,000 3286 tag SNPs; SNPs associated with breast, colorectal, lung, ovarian, and prostate cancers; SNPs 3287 covering ancestry, quantitative traits, and pharmacogenetics; and space for up to 120,000 custom 3288 SNPs. In collaboration with the National Cancer Institutes’ Genetic Association Mechanisms in 3289 Oncology (GAME-ON) consortium, the genotype data obtained will be submitted to the NIH 3290 database of Genotypes and Phenotypes (dbGAP) and will be available for subsequent sharing for 3291 research purposes. The identities of research participants will not be disclosed to dbGAP; the 3292 dataset will be de-identified. 3293 3294 14.16 Data Analyses 3295 Data is collected and stored at Geisel School of Medicine at Dartmouth. Per federal regulations, 3296 research using de-identified data does not involve "human subjects" and does not require IRB 3297 review. Thus, de-identified data from this data set may be sent outside of Geisel School of 3298 Medicine at Dartmouth for new analyses that are not specified in this protocol without additional 3299 IRB review. 3300 3301 3302 15.0 References 3303 3304 1. Landis SH, Murray T, Bolden S, Wingo PA. Cancer Statistics. CA Cancer J Clin 1998;48:6- 3305 29. 3306 2. Reichel H, Koeffler P, Norman A. The role of the vitamin D endocrine system in health and 3307 disease. N Engl J Med 1989;1989:980-91. 3308 3. Fraser DR. Vitamin D Lancet 1995;345:104-7. 3309 4. Lau KH, Baylink DJ. Vitamin D therapy of osteoporosis: plain vitamin D therapy versus 3310 active vitamin D analog (D-hormone) therapy. Calcified Tissue International 1999;65:295- 3311 306. 3312 5. Holick M. The use and interpretation of assays for vitamin D and its metabolites. J Nutr 3313 1990;120:1464-9. 3314 6. Brown AJ, Dusso A, Slatopolsky E. Vitamin D. American Journal of Physiology 3315 1999;277:F157-75.

v 20.0 9-11-14 72

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3316 7. Bell NH. Renal and nonrenal 25-hydroxyvitamin D-1alpha-hydroxylases and their clinical 3317 significance. Journal of Bone & Mineral Research 1998;13:350-3. 3318 8. Institute of Medicine. Standing Committee on the Scientific Evaluation of Dietary Reference 3319 Intakes. Food and Nutrition Board. In: Dietary Reference Intakes for Calcium, Phosphorus, 3320 Magnesium, Vitamin D and Fluoride. Washington, D.C.: National Academy Press; 1997:432. 3321 9. Barger-Lux MJ, Heaney RP, Lanspa SJ, Healy JC, DeLuca HF. An investigation of sources of 3322 variation in calcium absorption efficiency. Journal of Clinical Endocrinology & Metabolism 3323 1995;80:406-11. 3324 10. Heaney RP, Barger-Lux MJ, Dowell MS, Chen TC, Holick MF. Calcium absorptive 3325 effects of vitamin D and its major metabolites. Journal of Clinical Endocrinology & 3326 Metabolism 1997;82:4111-6. 3327 11. Holick MF. Vitamin D requirements for humans of all ages: new increased requirements 3328 for women and men 50 years and older. Osteoporosis International 1998;8:S24-9. 3329 12. Holick MF. Sunlight "D"ilemma: risk of skin cancer or bone disease and muscle 3330 weakness. Lancet 2001;357:4-6. 3331 13. Norman AW, Song X, Zanello L, Bula C, Okamura WH. Rapid and genomic biological 3332 responses are mediated by different shapes of the agonist steroid hormone, 3333 1alpha,25(OH)2vitamin D3. Steroids 1999;64:120-8. 3334 14. Curran JE, Vaughan T, Lea RA, Weinstein SR, Morrison NA, Griffiths LR. Association of 3335 A vitamin D receptor polymorphism with sporadic breast cancer development. International 3336 Journal of Cancer 1999;83:723-6. 3337 15. Dunning AM, McBride S, Gregory J, et al. No association between androgen or vitamin D 3338 receptor gene polymorphisms and risk of breast cancer. Carcinogenesis 1999;20:2131-5. 3339 16. Ingles SA, Ross RK, Yu MC, et al. Association of prostate cancer risk with genetic 3340 polymorphisms in vitamin D receptor and androgen receptor Journal of the National Cancer 3341 Institute 1997;89:166-70. 3342 17. Carling T, Ridefelt P, Hellman P, Rastad J, Akerstrom G. Vitamin D receptor 3343 polymorphisms correlate to parathyroid cell function in primary hyperparathyroidism. Journal 3344 of Clinical Endocrinology & Metabolism 1997;82:1772-5. 3345 18. Ingles SA, Haile RW, Henderson BE, et al. Strength of linkage disequilibrium between 3346 two vitamin D receptor markers in five ethnic groups: implications for association studies. 3347 Cancer Epidemiology, Biomarkers & Prevention 1997;6:93-8. 3348 19. Ingles SA, Garcia DG, Wang W, et al. Vitamin D receptor genotype and breast cancer in 3349 Latinas (United States). Cancer Causes & Control 2000;11:25-30. 3350 20. Ma J, Stampfer MJ, Gann PH, et al. Vitamin D receptor polymorphisms, circulating 3351 vitamin D metabolites, and risk of prostate cancer in United States physicians. Cancer 3352 Epidemiology, Biomarkers & Prevention 1998;7:385-90. 3353 21. Wishart JM, Horowitz M, Need AG, et al. Relations between calcium intake, calcitriol, 3354 polymorphisms of the vitamin D receptor gene, and calcium absorption in premenopausal 3355 women. American Journal of Clinical Nutrition 1997;65:798-802. 3356 22. Dawson-Hughes B, Harris SS, Finneran S. Calcium absorption on high and low calcium 3357 intakes in relation to vitamin D receptor genotype. Journal of Clinical Endocrinology & 3358 Metabolism 1995;80:3657-61. 3359 23. Campbell MJ, Koeffler HP. Toward therapeutic intervention of cancer by vitamin D 3360 compounds Journal of the National Cancer Institute 1997;89:182-5. 3361 24. Cross HS, Pavelka M, Slavik J, Peterlik M. Growth control of human colon cancer cells 3362 by vitamin D and calcium in vitro. Journal of the National Cancer Institute 1992;84:1355-7.

v 20.0 9-11-14 73

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3363 25. Lointier P, Wargovich MJ, Saez S, Levin B, Wildrick DM, Boman BM. The role of 3364 vitamin D3 in the proliferation of a human colon cancer cell line in vitro. Anticancer Research 3365 1987;7:817-21. 3366 26. James SY, Mackay AG, Colston KW. Effects of 1,25 dihydroxyvitamin D3 and its 3367 analogues on induction of apoptosis in breast cancer cells. Journal of Steroid Biochemistry & 3368 Molecular Biology 1996;58:395-401. 3369 27. Diaz GD, Paraskeva C, Thomas MG, Binderup L, Hague A. Apoptosis is induced by the 3370 active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and 3371 carcinoma cells: possible implications for prevention and therapy. Cancer Research 3372 2000;60:2304-12. 3373 28. Holick MF. 1,25-Dihydroxyvitamin D3 and the skin: a unique application for the 3374 treatment of psoriasis. Proceedings of the Society for Experimental Biology & Medicine 3375 1989;191:246-57. 3376 29. Holick MF. Noncalcemic actions of 1,25-dihydroxyvitamin D3 and clinical applications. 3377 Bone 1995;17:107S-11S. 3378 30. Menne T, Larsen K. Psoriasis treatment with vitamin D derivatives. Seminars in 3379 Dermatology 1992;11:278-83. 3380 31. Park BS, Park JS, Lee DY, Youn JI, Kim IG. Vitamin D receptor polymorphism is 3381 associated with psoriasis. Journal of Investigative Dermatology 1999;112:113-6. 3382 32. Thomas MG, Tebbutt S, Williamson RC. Vitamin D and its metabolites inhibit cell 3383 proliferation in human rectal mucosa and a colon cancer cell line. Gut 1992;33:1660-3. 3384 33. Thomas MG, Nugent KP, Forbes A, Williamson RC. Calcipotriol inhibits rectal epithelial 3385 cell proliferation in ulcerative proctocolitis. Gut 1994;35:1718-20. 3386 34. Sadava D, Remer T, Petersen K. Hyperplasia, hyperproliferation and decreased migration 3387 rate of colonic epithelial cells in mice fed a diet deficient in vitamin D. Biology of the Cell 3388 1996;87:113-5. 3389 35. Hashiba H, Fukushima M, Chida K, Kuroki T. Systemic inhibition of tumor promoter- 3390 induced ornithine decarboxylase in 1 alpha-hydroxyvitamin D3-treated animals. Cancer 3391 Research 1987;47:5031-5. 3392 36. Belleli A, Shany S, Levy J, Guberman R, Lamprecht SA. A protective role of 1,25- 3393 dihydroxyvitamin D3 in chemically induced rat colon carcinogenesis. Carcinogenesis 3394 1992;13:2293-8. 3395 37. Meggouh F, Lointier P, Saez S. Sex steroid and 1,25-dihydroxyvitamin D3 receptors in 3396 human colorectal adenocarcinoma and normal mucosa. Cancer Research 1991;51:1227-33. 3397 38. Vandewalle B, Adenis A, Hornez L, Revillion F, Lefebvre J. 1,25-dihydroxyvitamin D3 3398 receptors in normal and malignant human colorectal tissues. Cancer Letters 1994;86:67-73. 3399 39. Shabahang M, Buras RR, Davoodi F, Schumaker LM, Nauta RJ, Evans SR. 1,25- 3400 Dihydroxyvitamin D3 receptor as a marker of human colon carcinoma cell line differentiation 3401 and growth inhibition. Cancer Research 1993;53:3712-8. 3402 40. Evans SR, Nolla J, Hanfelt J, Shabahang M, Nauta RJ, Shchepotin IB. Vitamin D receptor 3403 expression as a predictive marker of biological behavior in human colorectal cancer. Clinical 3404 Cancer Research 1998;4:1591-5. 3405 41. Holt PR, Arber N, Halmos B, et al. Colonic epithelial cell proliferation decreases with 3406 increasing levels of serum 25-hydroxy vitamin D. Cancer Epidemiology, Biomarkers & 3407 Prevention 2002;11:113-9. 3408 42. Eisman JA, Barkla DH, Tutton PJ. Suppression of in vivo growth of human cancer solid 3409 tumor xenografts by 1,25-dihydroxyvitamin D3. Cancer Research 1987;47:21-5.

v 20.0 9-11-14 74

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3410 43. Sitrin MD, Halline AG, Abrahams C, Brasitus TA. Dietary calcium and vitamin D 3411 modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat. Cancer Research 3412 1991;51:5608-13. 3413 44. Huerta S, Irwin RW, Heber D, et al. 1alpha,25-(OH)(2)-D(3) and its synthetic analogue 3414 decrease tumor load in the Apc(min) Mouse. Cancer Research 2002;62:741-6. 3415 45. Kawaura A, Tanida N, Sawada K, Oda M, Shimoyama T. Supplemental administration of 3416 1 alpha-hydroxyvitamin D3 inhibits promotion by intrarectal instillation of lithocholic acid in 3417 N-methyl-N-nitrosourea-induced colonic tumorigenesis in rats. Carcinogenesis 1989;10:647- 3418 9. 3419 46. Beaty MM, Lee EY, Glauert HP. Influence of dietary calcium and vitamin D on colon 3420 epithelial cell proliferation and 1,2-dimethylhydrazine-induced colon carcinogenesis in rats 3421 fed high fat diets. Journal of Nutrition 1993;123:144-52. 3422 47. Evans SR, Shchepotin EI, Young H, Rochon J, Uskokovic M, Shchepotin IB. 1,25- 3423 dihydroxyvitamin D3 synthetic analogs inhibit spontaneous metastases in a 1,2- 3424 dimethylhydrazine-induced colon carcinogenesis model. International Journal of Oncology 3425 2000;16:1249-54. 3426 48. Comer PF, Clark TD, Glauert HP. Effect of dietary vitamin D3 (cholecalciferol) on colon 3427 carcinogenesis induced by 1,2-dimethylhydrazine in male Fischer 344 rats. Nutrition & 3428 Cancer 1993;19:113-24. 3429 49. van den Bemd GJ, Pols HA, van Leeuwen JP. Anti-tumor effects of 1,25- 3430 dihydroxyvitamin D3 and vitamin D analogs. Current Pharmaceutical Design 2000;6:717-32. 3431 50. Haussler MR, Whitfield GK, Haussler CA, et al. The nuclear vitamin D receptor: 3432 biological and molecular regulatory properties revealed. Journal of Bone & Mineral Research 3433 1998;13:325-49. 3434 51. Moffatt KA, Johannes WU, Hedlund TE, Miller GJ. Growth inhibitory effects of 1alpha, 3435 25-dihydroxyvitamin D(3) are mediated by increased levels of p21 in the prostatic carcinoma 3436 cell line ALVA-31. Cancer Research 2001;61:7122-9. 3437 52. Tong WM, Kallay E, Hofer H, et al. Growth regulation of human colon cancer cells by 3438 epidermal growth factor and 1,25-dihydroxyvitamin D3 is mediated by mutual modulation of 3439 receptor expression. European Journal of Cancer 1998;34:2119-25. 3440 53. Tong WM, Hofer H, Ellinger A, Peterlik M, Cross HS. Mechanism of antimitogenic 3441 action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal 3442 growth factor-stimulated cell growth. Oncology Research 1999;11:77-84. 3443 54. Xie SP, Pirianov G, Colston KW. Vitamin D analogues suppress IGF-I signalling and 3444 promote apoptosis in breast cancer cells. European Journal of Cancer 1999;35:1717-23. 3445 55. Salim EI, Wanibuchi H, Taniyama T, et al. Inhibition of development of N,N'- 3446 dimethylhydrazine-induced rat colonic aberrant crypt foci by pre, post and simultaneous 3447 treatments with 24R,25-dihydroxyvitamin D3. Japanese Journal of Cancer Research 3448 1997;88:1052-62. 3449 56. Makishima M, Lu TT, Xie W, et al. Vitamin D receptor as an intestinal bile acid sensor. 3450 Science 2002;296:1313-6. 3451 57. DeLuca HF, Zierold C. Mechanisms and functions of vitamin D. Nutrition Reviews 3452 1998;56:S4-10; discussion S 54-75. 3453 58. Lemire J. 1,25-Dihydroxyvitamin D3--a hormone with immunomodulatory properties. 3454 Zeitschrift fur Rheumatologie 2000;59:24-7. 3455 59. Garland CF, Garland FC. Do sunlight and vitamin D reduce the likelihood of colon 3456 cancer? International Journal of Epidemiology 1980;9:227-31.

v 20.0 9-11-14 75

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3457 60. Emerson JC, Weiss NS. Colorectal cancer and solar radiation. Cancer Causes & Control 3458 1992;3:95-9. 3459 61. Benito E, Stiggelbout A, Bosch FX, et al. Nutritional factors in colorectal cancer risk: a 3460 case-control study in Majorca. International Journal of Cancer 1991;49:161-7. 3461 62. Ferraroni M, La Vecchia C, D'Avanzo B, Negri E, Franceschi S, Decarli A. Selected 3462 micronutrient intake and the risk of colorectal cancer. British Journal of Cancer 3463 1994;70:1150-5. 3464 63. Pritchard RS, Baron JA, Gerhardsson de Verdier M. Dietary calcium, vitamin D, and the 3465 risk of colorectal cancer in Stockholm, Sweden. Cancer Epidemiology, Biomarkers & 3466 Prevention 1996;5:897-900. 3467 64. Kampman E, Slattery ML, Caan B, Potter JD. Calcium, vitamin D, sunshine exposure, 3468 dairy products and colon cancer risk (United States). Cancer Causes & Control 2000;11:459- 3469 66. 3470 65. Olsen J, Kronborg O, Lynggaard J, Ewertz M. Dietary risk factors for cancer and 3471 adenomas of the large intestine. A case-control study within a screening trial in Denmark. 3472 European Journal of Cancer 1994;30A:53-60. 3473 66. Marcus PM, Newcomb PA. The association of calcium and vitamin D, and colon and 3474 rectal cancer in Wisconsin women. International Journal of Epidemiology 1998;27:788-93. 3475 67. Breuer-Katschinski B, Nemes K, Marr A, et al. Colorectal adenomas and diet: a case- 3476 control study. Colorectal Adenoma Study Group. Digestive Diseases & Sciences 2001;46:86- 3477 95. 3478 68. Peters RK, Pike MC, Garabrant D, Mack TM. Diet and colon cancer in Los Angeles 3479 County, California. Cancer Causes & Control 1992;3:457-73. 3480 69. Boutron MC, Faivre J, Marteau P, Couillault C, Senesse P, Quipourt V. Calcium, 3481 phosphorus, vitamin D, dairy products and colorectal carcinogenesis: a French case--control 3482 study British Journal of Cancer 1996;74:145-51. 3483 70. Levine AJ, Harper JM, Ervin CM, et al. Serum 25-hydroxyvitamin D, dietary calcium 3484 intake, and distal colorectal adenoma risk. Nutrition & Cancer 2001;39:35-41. 3485 71. Benito E, Cabeza E, Moreno V, Obrador A, Bosch FX. Diet and colorectal adenomas: a 3486 case-control study in Majorca. International Journal of Cancer 1993;55:213-9. 3487 72. Little J, Logan RF, Hawtin PG, Hardcastle JD, Turner ID. Colorectal adenomas and diet: a 3488 case-control study of subjects participating in the Nottingham faecal occult blood screening 3489 programme. British Journal of Cancer 1993;67:177-84. 3490 73. Levi F, Pasche C, Lucchini F, La Vecchia C. Selected micronutrients and colorectal 3491 cancer. a case-control study from the canton of Vaud, Switzerland. European Journal of 3492 Cancer 2000;36:2115-9. 3493 74. Zheng W, Anderson KE, Kushi LH, et al. A prospective cohort study of intake of calcium, 3494 vitamin D, and other micronutrients in relation to incidence of rectal cancer among 3495 postmenopausal women. Cancer Epidemiology, Biomarkers & Prevention 1998;7:221-5. 3496 75. Martinez M, Giovannucci E, Colditz G, et al. Calcium, vitamin D, and the occurrence of 3497 colorectal cancer among women. Journal of the National Cancer Institute 1996;88:1375-82. 3498 76. Kearney J, Giovannucci E, Rimm EB, et al. Calcium, vitamin D, and dairy foods and the 3499 occurrence of colon cancer in men. American Journal of Epidemiology 1996;143:907-17. 3500 77. Bostick RM, Potter JD, Sellers TA, McKenzie DR, Kushi LH, Folsom AR. Relation of 3501 calcium, vitamin D, and dairy food intake to incidence of colon cancer among older women. 3502 The Iowa Women's Health Study. American Journal of Epidemiology 1993;137:1302-17.

v 20.0 9-11-14 76

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3503 78. Garland C, Shekelle RB, Barrett-Connor E, Criqui MH, Rossof AH, Paul O. Dietary 3504 vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study in men. 3505 Lancet 1985;1:307-9. 3506 79. Jarvinen R, Knekt P, T. H, Aromaa A. Prospective study on milk products, calcium and 3507 cancers of the colon and rectum. European Journal of Clinical Nutrition 2000;55:1000-7. 3508 80. Garland C, Comstock G, Garland F, Helsing K, Shaw E, Gorham E. Serum 35- 3509 hydroxyvitamin D and colon cancer: eight year prospective study. Lancet 1989;ii:1176-9. 3510 81. Braun MM, Helzlsouer KJ, Hollis BW, Comstock GW. Colon cancer and serum vitamin 3511 D metabolite levels 10-17 years prior to diagnosis. American Journal of Epidemiology 3512 1995;142:608-11. 3513 82. Tangrea J, Helzlsouer K, Pietinen P, et al. Serum levels of vitamin D metabolites and the 3514 subsequent risk of colon and rectal cancer in Finnish men. Cancer Causes & Control 3515 1997;8:615-25. 3516 83. Peters U, McGlynn KA, Chatterjee N, et al. Vitamin d, calcium, and vitamin d receptor 3517 polymorphism in colorectal adenomas. Cancer Epidemiology, Biomarkers & Prevention 3518 2001;10:1267-74. 3519 84. Slattery ML, Yakumo K, Hoffman M, Neuhausen S. Variants of the VDR gene and risk of 3520 colon cancer (United States). Cancer Causes & Control 2001;12:359-64. 3521 85. Ingles SA, Wang J, Coetzee GA, Lee ER, Frankl HD, Haile RW. Vitamin D receptor 3522 polymorphisms and risk of colorectal adenomas (United States). Cancer Causes & Control 3523 2001;12:607-14. 3524 86. Kim HS, Newcomb PA, Ulrich CM, et al. Vitamin D receptor polymorphism and the risk 3525 of colorectal adenomas: evidence of interaction with dietary vitamin D and calcium. Cancer 3526 Epidemiology, Biomarkers & Prevention 2001;10:869-74. 3527 87. Tangpricha V, Flanagan JN, Whitlatch LW, et al. 25-hydroxyvitamin D-1alpha- 3528 hydroxylase in normal and malignant colon tissue. Lancet 2001;357:1673-4. 3529 88. Cross HS, Bareis P, Hofer H, et al. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase and 3530 vitamin D receptor gene expression in human colonic mucosa is elevated during early 3531 cancerogenesis. Steroids 2001;66:287-92. 3532 89. Cross HS, Hulla W, Tong WM, Peterlik M. Growth regulation of human colon 3533 adenocarcinoma-derived cells by calcium, vitamin D and epidermal growth factor. Journal of 3534 Nutrition 1995;125:2004S-8S. 3535 90. Cross HS, Peterlik M, Reddy GS, Schuster I. Vitamin D metabolism in human colon 3536 adenocarcinoma-derived Caco-2 cells: expression of 25-hydroxyvitamin D3-1alpha- 3537 hydroxylase activity and regulation of side-chain metabolism. Journal of Steroid Biochemistry 3538 & Molecular Biology 1997;62:21-8. 3539 91. Schwartz GG, Whitlatch LW, Chen TC, Lokeshwar BL, Holick MF. Human prostate cells 3540 synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. Cancer Epidemiology, 3541 Biomarkers & Prevention 1998;7:391-5. 3542 92. Barreto AM, Schwartz GG, Woodruff R, Cramer SD. 25-Hydroxyvitamin D3, the 3543 prohormone of 1,25-dihydroxyvitamin D3, inhibits the proliferation of primary prostatic 3544 epithelial cells. Cancer Epidemiology, Biomarkers & Prevention 2000;9:265-70. 3545 93. Chen TC, Schwartz GG, Burnstein KL, Lokeshwar BL, Holick MF. The in vitro 3546 evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as 3547 therapeutic agents for prostate cancer. Clinical Cancer Research 2000;6:901-8. 3548 94. Institute of Medicine. Dietary reference intakes for calcium and vitamin D. Washington, 3549 DC: National Academies Press, 2011.

v 20.0 9-11-14 77

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3550 95. van der Wielen RP, Lowik MR, van den Berg H, et al. Serum vitamin D concentrations 3551 among elderly people in Europe. Lancet 1995;346:207-10. 3552 96. Vieth R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety 3553 American Journal of Clinical Nutrition 1999;69:842-56. 3554 97. Chapuy MC, Preziosi P, Maamer M, et al. Prevalence of vitamin D insufficiency in an 3555 adult normal population. Osteoporosis International 1997;7:439-43. 3556 98. Malabanan A, Veronikis IE, Holick MF. Redefining vitamin D insufficiency Lancet 3557 1998;351:805-6. 3558 99. Gloth FM, 3rd, Tobin JD. Vitamin D deficiency in older people. Journal of the American 3559 Geriatrics Society 1995;43:822-8. 3560 100. Byrne PM, Freaney R, McKenna MJ. Vitamin D supplementation in the elderly: review of 3561 safety and effectiveness of different regimes. Calcified Tissue International 1995;56:518-20. 3562 101. Harris SS, Dawson-Hughes B. Plasma Vitamin D and 25OHD responses of young and old 3563 men to supplementation with Vitamin D3. Journal of the American College of Nutrition 3564 2002;21:357-62. 3565 102. Chapuy MC, Chapuy P, Thomas JL, Hazard MC, Meunier PJ. Biochemical effects of 3566 calcium and vitamin D supplementation in elderly, institutionalized, vitamin D-deficient 3567 patients. Revue du Rhumatisme (English Edition) 1996;63:135-40. 3568 103. Sebert JL, Garabedian M, Chauvenet M, Maamer M, Agbomson F, Brazier M. Evaluation 3569 of a new solid formulation of calcium and vitamin D in institutionalized elderly subjects. A 3570 randomized comparative trial versus separate administration of both constituents. Revue Du 3571 Rhumatisme, English Edition 1995;62:288-94. 3572 104. Himmelstein S, Clemens TL, Rubin A, Lindsay R. Vitamin D supplementation in elderly 3573 nursing home residents increases 25(OH)D but not 1,25(OH)2D. American Journal of Clinical 3574 Nutrition 1990;52:701-6. 3575 105. Patel R, Collins D, Bullock S, Swaminathan R, Blake GM, Fogelman I. The effect of 3576 season and vitamin D supplementation on bone mineral density in healthy women: a double- 3577 masked crossover study. Osteoporosis International 2001;12:319-25. 3578 106. Lips P, Wiersinga A, van Ginkel FC, et al. The effect of vitamin D supplementation on 3579 vitamin D status and parathyroid function in elderly subjects. Journal of Clinical 3580 Endocrinology & Metabolism 1988;67:644-50. 3581 107. Barger-Lux MJ, Heaney RP, Dowell S, Chen TC, Holick MF. Vitamin D and its major 3582 metabolites: serum levels after graded oral dosing in healthy men. Osteoporosis International 3583 1998;8:222-30. 3584 108. Honkanen R, Alhava E, Parviainen M, Talasniemi S, Monkkonen R. The necessity and 3585 safety of calcium and vitamin D in the elderly. Journal of the American Geriatrics Society 3586 1990;38:862-6. 3587 109. Chapuy MC, Meunier PJ. Prevention of secondary hyperparathyroidism and hip fracture 3588 in elderly women with calcium and vitamin D3 supplements. Osteoporosis International 3589 1996;6:60-3. 3590 110. MacLennan WJ, Hamilton JC. Vitamin D supplements and 25-hydroxy vitamin D 3591 concentrations in the elderly. Bmj 1977;2:859-61. 3592 111. Chapuy MC, Chapuy P, Meunier PJ. Calcium and vitamin D supplements: effects on 3593 calcium metabolism in elderly people. American Journal of Clinical Nutrition 1987;46:324-8. 3594 112. Chapuy MC, Arlot ME, Duboeuf F, et al. Vitamin D3 and calcium to prevent hip fractures 3595 in the elderly women. New England Journal of Medicine 1992;327:1637-42.

v 20.0 9-11-14 78

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3596 113. Sorva A, Risteli J, Risteli L, Valimaki M, Tilvis R. Effects of vitamin D and calcium on 3597 markers of bone metabolism in geriatric patients with low serum 25-hydroxyvitamin D levels. 3598 Calcified Tissue International 1991;49:S88-9. 3599 114. Vieth R, Chan PC, MacFarlane GD. Efficacy and safety of vitamin D(3) intake exceeding 3600 the lowest observed adverse effect level. American Journal of Clinical Nutrition 2001;73:288- 3601 94. 3602 115. Gannage-Yared MH, Chemali R, Yaacoub N, Halaby G. Hypovitaminosis D in a sunny 3603 country: relation to lifestyle and bone markers. Journal of Bone & Mineral Research 3604 2000;15:1856-62. 3605 116. Goswami R, Gupta N, Goswami D, Marwaha RK, Tandon N, Kochupillai N. Prevalence 3606 and significance of low 25-hydroxyvitamin D concentrations in healthy subjects in Delhi. 3607 American Journal of Clinical Nutrition 2000;72:472-5. 3608 117. Thomas MK, Lloyd-Jones DM, Thadhani RI, et al. Hypovitaminosis D in medical 3609 inpatients New England Journal of Medicine 1998;338:777-83. 3610 118. Semba RD, Garrett E, Johnson BA, Guralnik JM, Fried LP. Vitamin D deficiency among 3611 older women with and without disability. American Journal of Clinical Nutrition 3612 2000;72:1529-34. 3613 119. Nagengast FM, Grubben MJ, van Munster IP. Role of bile acids in colorectal 3614 carcinogenesis. European Journal of Cancer 1995;31A:1067-70. 3615 120. McSherry C, Cohen B, Bokkenheuser V, et al. Effects of calcium and bile acid feeding on 3616 colon tumors in the rat. Cancer Res 1989;49:6039-43. 3617 121. Galloway DJ, Owen RW, Jarrett F, Boyle P, Hill MJ, George WD. Experimental 3618 colorectal cancer: the relationship of diet and faecal bile acid concentration to tumour 3619 induction. British Journal of Surgery 1986;73:233-7. 3620 122. Hill MJ. Bile flow and colon cancer. Mutation Research 1990;238:313-20. 3621 123. Hill MJ. Bile acids and colorectal cancer: hypothesis. European Journal of Cancer 3622 Prevention 1991;1:69-74. 3623 124. Newmark HL, Wargovich MJ, Bruce WR. Colon cancer and dietary fat, phosphate, and 3624 calcium: a hypothesis. Journal of the National Cancer Institute 1984;72:1323-5. 3625 125. Van der Meer R, Lapre JA, Govers MJ, Kleibeuker JH. Mechanisms of the intestinal 3626 effects of dietary fats and milk products on colon carcinogenesis. Cancer Letters 1997;114:75- 3627 83. 3628 126. Kallay E, Bajna E, Wrba F, Kriwanek S, Peterlik M, Cross HS. Dietary calcium and 3629 growth modulation of human colon cancer cells: role of the extracellular calcium-sensing 3630 receptor. Cancer Detection & Prevention 2000;24:127-36. 3631 127. Kallay E, Kifor O, Chattopadhyay N, et al. Calcium-dependent c-myc proto-oncogene 3632 expression and proliferation of Caco-2 cells: a role for a luminal extracellular calcium-sensing 3633 receptor. Biochemical & Biophysical Research Communications 1997;232:80-3. 3634 128. Reshef R, Rozen P, Fireman Z, et al. Effect of a calcium-enriched diet on the colonic 3635 epithelial hyperproliferation induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats on a 3636 low calcium and fat diet. Cancer Res 1990;50:1764-7. 3637 129. Nobre-Leitao C, Chaves P, Fidalgo P, et al. Calcium regulation of coloic crypt cell 3638 kinetics: evidence for a direct effect in mice. Gastroenterology 1995;109:498-504. 3639 130. Wargovich MJ, Eng VW, Newmark HL, Bruce WR. Calcium ameliorates the toxic effect 3640 of deoxycholic acid on colonic epithelium. Carcinogenesis 1983;4:1205-7. 3641 131. Bird RP, Schneider R, Stamp D, Bruce WR. Effect of dietary calcium and cholic acid on 3642 the proliferative indices of murine colonic epithelium. Carcinogenesis 1986;7:1657-61.

v 20.0 9-11-14 79

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3643 132. Appleton GV, Bristol JB, Williamson RC. Increased dietary calcium and small bowel 3644 resection have opposite effects on colonic cell turnover. British Journal of Surgery 3645 1986;73:1018-21. 3646 133. Appleton GV, Davies PW, Bristol JB, Williamson RC. Inhibition of intestinal 3647 carcinogenesis by dietary supplementation with calcium. British Journal of Surgery 3648 1987;74:523-5. 3649 134. Pence BC, Buddingh F. Inhibition of dietary fat-promoted colon carcinogenesis in rats by 3650 supplemental calcium or vitamin D3. Carcinogenesis 1988;9:187-90. 3651 135. Wargovich M, Allnutt D, Palmer C, Anaya P, Stephens L. Inhibition of the promotional 3652 phase of azoxymethane-induced colon carcinogenesis in the rat by calcium lactate: effect of 3653 simulating two human nutrient density levels. Cancer Lett 1990;53:17-25. 3654 136. Pence BC, Dunn DM, Zhao C, Landers M, Wargovich MJ. Chemopreventive effects of 3655 calcium but not aspirin supplementation in cholic acid-promoted colon carcinogenesis: 3656 correlation with intermediate endpoints. Carcinogenesis 1995;16:757-65. 3657 137. Karkare M, Clark T, Glauert H. Effect of dietary calcium on colon carcinogenesis induced 3658 by a single injection of 1,2-dimethylhydrazine in rats. J Nutr 1991;121:568-77. 3659 138. Wu K, Willett WC, Fuchs CS, Colditz GA, Giovannucci EL. Calcium intake and risk of 3660 colon cancer in women and men. Journal of the National Cancer Institute 2002;94:437-46. 3661 139. Heilbrun L, Hankin J, Nomura A, Stemmermann G. Colon cancer and dietary fat; 3662 phosphorus; and calcium in Hawaiian-Japanese men. Am J Clin Nutr 1986;1986:306-9. 3663 140. Kampman E, Goldbohm R, Van Den Brandt P, Van't Veer P. Fermented dairy products, 3664 calcium, and colorectal cancer in the Netherlands cohort study. Can Res 1994;54:3186-90. 3665 141. Bergsma-Kadijk JA, van 't Veer P, Kampman E, Burema J. Calcium does not protect 3666 against colorectal neoplasia. Epidemiology 1996;7:590-7. 3667 142. Martinez ME, Willett WC. Calcium, Vitamin D, and Colorectal Cancer: A Review of the 3668 Epidemiologic Evidence. Cancer Epidemiology, Biomarkers & Prevention 1998;7:163-8. 3669 143. Slattery ML, Sorenson AW, Ford MH. Dietary calcium intake as a mitigating factor in 3670 colon cancer. American Journal of Epidemiology 1988;128:504-14. 3671 144. Kampman E, Giovannucci E, van 't Veer P, et al. Calcium, vitamin D, dairy foods, and the 3672 occurrence of colorectal adenomas among men and women in two prospective studies. 3673 American Journal of Epidemiology 1994;139:16-29. 3674 145. Macquart-Moulin G, Riboli E, Cornee J, Kaaks R, Berthezene P. Colorectal polyps and 3675 diet: a case-control study in Marseilles. Int J Cancer 1987;40:179-88. 3676 146. Tseng M, Murray SC, Kupper LL, Sandler RS. Micronutrients and the risk of colorectal 3677 adenomas American Journal of Epidemiology 1996;144:1005-14. 3678 147. Hyman J, Baron JA, Dain BJ, et al. Dietary and supplemental calcium and the recurrence 3679 of colorectal adenomas. Cancer Epidemiology, Biomarkers & Prevention 1998;7:291-5. 3680 148. Almendingen K, Trygg K, Larsen S, Hofstad B, Vatn MH. Dietary factors and colorectal 3681 polyps: a case-control study. European Journal of Cancer Prevention 1995;4:239-46. 3682 149. Neugut A, Horvath K, Whelan R, et al. The effect of calcium and vitamin supplements on 3683 the incidence and recurrence of colorectal adenomatous polyps. Cancer 1996;78:723-8. 3684 150. Peleg, II, Lubin MF, Cotsonis GA, Clark WS, Wilcox CM. Long-term use of nonsteroidal 3685 antiinflammatory drugs and other chemopreventors and risk of subsequent colorectal 3686 neoplasia. Digestive Diseases & Sciences 1996;41:1319-26. 3687 151. White E, Shannon JS, Patterson RE. Relationship between vitamin and calcium 3688 supplement use and colon cancer. Cancer Epidemiology, Biomarkers & Prevention 3689 1997;6:769-74.

v 20.0 9-11-14 80

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3690 152. Whelan RL, Horvath KD, Gleason NR, et al. Vitamin and calcium supplement use is 3691 associated with decreased adenoma recurrence in patients with a previous history of 3692 neoplasia. Diseases of the Colon & Rectum 1999;42:212-7. 3693 153. Bonithon-Kopp C, Kronborg O, Giacosa A, Rath U, Faivre J. Calcium and fibre 3694 supplementation in prevention of colorectal adenoma recurrence: a randomised intervention 3695 trial. European Cancer Prevention Organisation Study Group. Lancet 2000;356:1300-6. 3696 154. Thomas MG, Thomson JP, Williamson RC. Oral calcium inhibits rectal epithelial 3697 proliferation in familial adenomatous polyposis. British Journal of Surgery 1993;80:499-501. 3698 155. Hofstad B, Almendingen K, Vatn M, et al. Growth and recurrence of colorectal polyps: a 3699 double-blind 3-year intervention with calcium and antioxidants. Digestion 1998;59:148-56. 3700 156. Gregoire RC, Stern HS, Yeung KS, et al. Effect of calcium supplementation on mucosal 3701 cell proliferation in high risk patients for colon cancer. Gut 1989;30:376-82. 3702 157. Stern HS, Gregoire RC, Kashtan H, Stadler J, Bruce RW. Long-term effects of dietary 3703 calcium on risk markers for colon cancer in patients with familial polyposis. Surgery 3704 1990;108:528-33. 3705 158. Bostick RM, Potter JD, Fosdick L, et al. Calcium and colorectal epithelial cell 3706 proliferation: a preliminary randomized, double-blinded, placebo-controlled clinical trial. 3707 Journal of the National Cancer Institute 1993;85:132-41. 3708 159. Kleibeuker JH, Welberg JW, Mulder NH, et al. Epithelial cell proliferation in the sigmoid 3709 colon of patients with adenomatous polyps increases during oral calcium supplementation. 3710 British Journal of Cancer 1993;67:500-3. 3711 160. Cats A, Kleibeuker JH, van der Meer R, et al. Randomized, double-blinded, placebo- 3712 controlled intervention study with supplemental calcium in families with hereditary 3713 nonpolyposis colorectal cancer. Journal of the National Cancer Institute 1995;87:598-603. 3714 161. Baron JA, Tosteson TD, Wargovich MJ, et al. Calcium supplementation and rectal 3715 mucosal proliferation: a randomized controlled trial Journal of the National Cancer Institute 3716 1995;87:1303-7. 3717 162. Armitage NC, Rooney PS, Gifford KA, Clarke PA, Hardcastle JD. The effect of calcium 3718 supplements on rectal mucosal proliferation. British Journal of Cancer 1995;71:186-90. 3719 163. Lipkin M, Newmark H. Effect of added dietary calcium on colonic epithelial-cell 3720 proliferation in subjects at high risk for familial colonic cancer. New England Journal of 3721 Medicine 1985;313:1381-4. 3722 164. Lipkin M, Friedman E, Winawer SJ, Newmark H. Colonic epithelial cell proliferation in 3723 responders and nonresponders to supplemental dietary calcium. Cancer Research 3724 1989;49:248-54. 3725 165. Rozen P, Fireman Z, Fine N, Wax Y, Ron E. Oral calcium suppresses increased rectal 3726 epithelial proliferation of persons at risk of colorectal cancer. Gut 1989;30:650-5. 3727 166. Barsoum GH, Hendrickse C, Winslet MC, et al. Reduction of mucosal crypt cell 3728 proliferation in patients with colorectal adenomatous polyps by dietary calcium 3729 supplementation. British Journal of Surgery 1992;79:581-3. 3730 167. Wargovich MJ, Isbell G, Shabot M, et al. Calcium supplementation decreases rectal 3731 epithelial cell proliferation in subjects with sporadic adenoma Gastroenterology 1992;103:92- 3732 7. 3733 168. Bostick RM, Fosdick L, Wood JR, et al. Calcium and colorectal epithelial cell 3734 proliferation in sporadic adenoma patients: a randomized, double-blinded, placebo-controlled 3735 clinical trial. Journal of the National Cancer Institute 1995;87:1307-15.

v 20.0 9-11-14 81

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3736 169. Lapre JA, De Vries HT, Termont DS, Kleibeuker JH, De Vries EG, Van der Meer R. 3737 Mechanism of the protective effect of supplemental dietary calcium on cytolytic activity of 3738 fecal water. Cancer Research 1993;53:248-53. 3739 170. Welberg JW, Kleibeuker JH, Van der Meer R, et al. Effects of oral calcium 3740 supplementation on intestinal bile acids and cytolytic activity of fecal water in patients with 3741 adenomatous polyps of the colon. European Journal of Clinical Investigation 1993;23:63-8. 3742 171. Lupton JR, Steinbach G, Chang WC, et al. Calcium supplementation modifies the relative 3743 amounts of bile acids in bile and affects key aspects of human colon physiology. Journal of 3744 Nutrition 1996;126:1421-8. 3745 172. Alder RJ, McKeown-Eyssen G, Bright-See E. Randomized trial of the effect of calcium 3746 supplementation on fecal risk factors for colorectal cancer. American Journal of 3747 Epidemiology 1993;138:804-14. 3748 173. Curhan GC, Willett WC, Rimm EB, Stampfer MJ. A prospective study of dietary calcium 3749 and other nutrients and the risk of symptomatic kidney stones. New England Journal of 3750 Medicine 1993;328:833-8. 3751 174. Curhan GC, Willett WC, Speizer FE, Spiegelman D, Stampfer MJ. Comparison of dietary 3752 calcium with supplemental calcium and other nutrients as factors affecting the risk for kidney 3753 stones in women. Annals of Internal Medicine 1997;126:497-504. 3754 175. Schwarz S, Trivedi BK, Kalantar-Zadeh K, Kovesdy CP. Association of disorders in 3755 mineral metabolism with progression of chronic kidney disease. Clin J Am Soc Nephrol 3756 2006;1:825-31. 3757 176. Melamed ML, Astor B, Michos ED, Hostetter TH, Powe NR, Muntner P. 25- 3758 hydroxyvitamin D levels, race, and the progression of kidney disease. J Am Soc Nephrol 3759 2009;20:2631-9. 3760 177. Ix JH, Shlipak MG, Wassel CL, Whooley MA. Fibroblast growth factor-23 and early 3761 decrements in kidney function: the Heart and Soul Study. Nephrol Dial Transplant 3762 2010;25:993-7. 3763 178. Li YC, Qiao G, Uskokovic M, Xiang W, Zheng W, Kong J. Vitamin D: a negative 3764 endocrine regulator of the renin-angiotensin system and blood pressure. J Steroid Biochem 3765 Mol Biol 2004;89-90:387-92. 3766 179. Migliori M, Giovannini L, Panichi V, et al. Treatment with 1,25-dihydroxyvitamin D3 3767 preserves glomerular slit diaphragm-associated protein expression in experimental 3768 glomerulonephritis. Int J Immunopathol Pharmacol 2005;18:779-90. 3769 180. Makibayashi K, Tatematsu M, Hirata M, et al. A vitamin D analog ameliorates glomerular 3770 injury on rat glomerulonephritis. Am J Pathol 2001;158:1733-41. 3771 181. Neves KR, Graciolli FG, dos Reis LM, Pasqualucci CA, Moyses RM, Jorgetti V. Adverse 3772 effects of hyperphosphatemia on myocardial hypertrophy, renal function, and bone in rats 3773 with renal failure. Kidney Int 2004;66:2237-44. 3774 182. Forman JP, Giovannucci E, Holmes MD, et al. Plasma 25-hydroxyvitamin D levels and 3775 risk of incident hypertension. Hypertension 2007;49:1063-9. 3776 183. Krause R, Buhring M, Hopfenmuller W, Holick MF, Sharma AM. Ultraviolet B and blood 3777 pressure. Lancet 1998;352:709-10. 3778 184. Pfeifer M, Begerow B, Minne HW, Nachtigall D, Hansen C. Effects of a short-term 3779 vitamin D(3) and calcium supplementation on blood pressure and parathyroid hormone levels 3780 in elderly women. J Clin Endocrinol Metab 2001;86:1633-7. 3781 185. Sugden JA, Davies JI, Witham MD, Morris AD, Struthers AD. Vitamin D improves 3782 endothelial function in patients with Type 2 diabetes mellitus and low vitamin D levels. 3783 Diabet Med 2008;25:320-5.

v 20.0 9-11-14 82

Downloaded From: https://jamanetwork.com/ on 09/26/2021 3784 186. Foster MC, Hwang SJ, Larson MG, et al. Cross-classification of microalbuminuria and 3785 reduced glomerular filtration rate: associations between cardiovascular disease risk factors 3786 and clinical outcomes. Arch Intern Med 2007;167:1386-92. 3787 187. Astor BC, Hallan SI, Miller ER, 3rd, Yeung E, Coresh J. Glomerular filtration rate, 3788 albuminuria, and risk of cardiovascular and all-cause mortality in the US population. 3789 American journal of epidemiology 2008;167:1226-34. 3790 188. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular 3791 filtration rate. Annals of internal medicine 2009;150:604-12. 3792 189. Pocock SJ, Hughes MD. Practical problems in interim analyses, with particular regard to 3793 estimation. Controlled Clinical Trials 1989;10:209S-21S. 3794 190. O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. Biometrics 3795 1979;35:549-56. 3796 3797

v 20.0 9-11-14 83

Downloaded From: https://jamanetwork.com/ on 09/26/2021