Trial Design and Oversight

We conducted this randomized, double-blind, placebo-controlled trial, with a two-by-two factorial design, to examine the benefits and risks of vitamin D 3 (cholecalciferol) at a dose of 2000 IU per day and marine n−3 fatty acids at a dose of 1 g per day in the primary prevention of cancer and cardiovascular disease among 25,871 men who were 50 years of age or older and women who were 55 years of age or older. The trial protocol has been described elsewhere4,12 and is available with the full text of this article at NEJM.org.

Participants were recruited throughout the United States, and the groups were balanced according to sex and with a goal to include at least 5000 black participants. Eligible participants had no history of cancer (except nonmelanoma skin cancer) or cardiovascular disease at trial entry, and they were required to agree to limit the use of vitamin D from all supplemental sources, including multivitamins, to 800 IU per day and to complete a 3-month placebo run-in phase. Safety exclusions included renal failure or dialysis, cirrhosis, history of hypercalcemia, or other serious conditions that would preclude participation. Randomization was computer generated within sex, race, and 5-year age groups in blocks of eight.

Baseline questionnaires collected data on risk factors for cancer, cardiovascular disease, and other conditions and included a food frequency questionnaire. Participants received follow-up questionnaires at 6 months and 1 year after randomization and annually thereafter to collect information on adherence to trial regimens, outside use of vitamin D supplements, development of major illnesses, updates on risk factors, and potential side effects of the trial agents. Calendar packs containing the trial capsules of vitamin D or corresponding placebo (and n−3 fatty acids or corresponding placebo) were mailed with questionnaires to the participants.

Blood samples were obtained at baseline during the run-in period from all willing participants — 16,956 of the 25,871 persons who underwent randomization (65.5%). At no cost to the trial, Quest Diagnostics donated and performed serum 25-hydroxyvitamin D assays with the use of liquid chromatography–tandem mass spectrometry on all samples that could be analyzed. Quest had no role in the design of the trial, accrual of the data (other than the assays), analysis of the data (other than assay standards), or preparation of the manuscript. Our trial participated in the vitamin D standardization program of the Centers for Disease Control and Prevention.13

The National Institutes of Health, the sponsors of the trial, had a collaborative role in the design and conduct of the trial. Final decisions regarding the data collection, management, and analysis and the review and approval of the manuscript and decision to submit the manuscript for publication resided with trial investigators and the trial research group. The trial was approved by the institutional review board of Partners HealthCare–Brigham and Women’s Hospital and was monitored by an external data and safety monitoring board. The trial agents have received Investigational New Drug Approval from the Food and Drug Administration. Pharmavite donated vitamin D and Pronova BioPharma and BASF donated fish oil (Omacor); the companies also donated matching placebos and packaging in the form of calendar packs. None of the donating companies had any role in the design or conduct of the trial, collection or analysis of the data, or preparation or review of the manuscript. The first three authors and the last author had full access to all the trial data and vouch for the completeness and accuracy of the data, for the accuracy of the data analyses, and for the fidelity of the trial to the protocol. All the participants provided written informed consent before enrollment in the trial.

Trial End Points

The primary end points were invasive cancer of any type and major cardiovascular events (composite of myocardial infarction, stroke, and death from cardiovascular causes). Secondary cancer end points were incident colorectal, breast, and prostate cancers, and death from cancer. Secondary cardiovascular end points were an expanded composite of major cardiovascular events plus coronary revascularization and the individual components of major cardiovascular events. Participants who reported an end-point event were asked to sign a release for medical records, which were reviewed for confirmation by an end-points committee of physicians who were unaware of the trial-group assignments. Cancer was confirmed on the basis of histologic or cytologic data.14 Myocardial infarction and stroke were confirmed with the use of established criteria,15,16 coronary revascularization was confirmed by medical record review, and death from cardiovascular causes was confirmed if there was convincing evidence of a cardiovascular event from all available sources. Analyses included only confirmed end points.

For deaths reported by family members, the next of kin was asked for permission to obtain medical records and a copy of the death certificate. Alternatively, the latter was obtained from the state vital records bureau. The end-points committee reviewed the records to assign the cause of death. If records were unavailable (or participants were lost to follow-up), the National Death Index (NDI) Plus was searched for cause of death according to the death-certificate information. Deaths were defined with the use of all these sources; a secondary analysis of cause-specific deaths required medical records or other adjudication of cause of death beyond NDI coding.

Statistical Analysis

Analyses of effect were based on the intention-to-treat principle (all participants who underwent randomization were included). The trial was designed to have a greater than 85% power to detect observed hazard ratios of 0.85 and 0.80 for the primary end points of cancer and cardiovascular disease, respectively.4 Initial analyses compared baseline characteristics of participants according to trial regimen with the use of t-tests or chi-square tests. Primary analyses compared the main effects of vitamin D on cancer and cardiovascular disease with the use of Cox proportional-hazards models that were controlled for age, sex, and randomization group in the n−3 fatty acid portion of the trial (n−3 fatty acid group or placebo group). Person-time was counted from randomization to the end point, to death, or to the end of the trial on December 31, 2017. Cumulative-incidence plots and interactions with time were used to examine whether effects varied over time. Prespecified analyses of the primary outcomes excluding events that occurred during the first year and the first 2 years of follow-up assessed latent effects. Adherence effects were estimated by censoring follow-up data when the participant discontinued trial capsules or began taking more than 800 IU per day of outside vitamin D.

Possible variations in the effect according to race or ethnic group, age, sex, body-mass index (BMI, the weight in kilograms divided by the square of the height in meters), baseline 25-hydroxyvitamin D level, concurrent randomization to the n−3 group, outside use of vitamin D supplements, and baseline risk factors for cancer and cardiovascular disease were specified a priori. However, there was no control for multiple hypothesis testing, and no formal adjustment was made to the P values or confidence intervals. Thus, results regarding secondary and exploratory end points, as well as those regarding subgroups, should be interpreted with caution. The incidence of potential side effects according to randomly assigned group was also compared.