Affiliation of authors: Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY.
Correspondence to: Anna J. Duffield-Lillico, PhD, Epidemiology Service, Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, 307 E. 63rd St., Third Floor, New York, NY 10021 (e-mail: lillicoa{at}mskcc.org)
The cancer prevention community was stunned in the early 1990s, when the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Trial, a randomized, 2 x 2 factorial prevention trial of daily 50 mg -tocopherol and/or 20 mg
-carotene conducted on more than 29 000 male smokers, was stopped ahead of schedule after results showed that subjects who received
-carotene had a statistically significant increase in lung cancer incidence compared with subjects who received placebo (1). The ATBC study was designed to test whether
-carotene could reduce the risk of lung cancer, a hypothesis that was based on substantial evidence from observational epidemiologic studies (24). Further impetus to terminate the ATBC study came from the observation that subjects who received
-carotene had a statistically significant increase in overall mortality compared with subjects who received placebo. Shortly after the ATBC study was terminated, the Carotene and Retinol Efficacy (CARET) Trial, a randomized study of the combination of daily 30 mg of
-carotene and 25 000 IU retinol versus placebo in individuals with a history of smoking or asbestos exposure, was also stopped ahead of schedule for exactly the same reasons, statistically significant excess incidences of lung cancer and overall mortality (5).
Large trials such as these require a tremendous investment of scarce resources. Ethical considerations require that a study be terminated when strong evidence emerges of a difference in efficacy between the study arms. A study cannot continue until the evidence is overwhelming. Thus, investigators are always anxious that subsequent follow-up of participants might ultimately show that a result that seemed compelling when the study was terminated is diminished or reversed on subsequent follow-up. The possibility that the adverse effects of -carotene observed in the CARET and ATBC trials might be false positives has certainly been an open question, especially because results from a third large prevention trial of
-carotene, the Physicians Health Study (PHS), showed no adverse effects on either endpoint (6). However, the PHS differed from the ATBC and CARET studies in two important ways: the PHS was not restricted to smokers, although 50% of the participants were either current (11%) or former (39%) smokers; and in the PHS, treatment with 50 mg of
-carotene every other day yielded a median blood level of
-carotene of 1.2 µg/mL, which was considerably lower than the median blood levels achieved in subjects who received the
-carotenecontaining supplements in the ATBC study and CARET (median blood levels of
-carotene: 3.0 µg/mL and 2.1 µg/mL, respectively). In addition, two smaller studies conducted in the United States have reported no effect of
-carotene with respect to cardiovascular, cancer, and mortality endpoints: the Women's Health Study, in which participants (13% of whom were smokers) were treated with 50 mg of
-carotene every other day for a mean period of 2.1 years (7), and the Skin Cancer Prevention Study, a randomized, placebo-controlled trial of 50 mg of
-carotene per day among 1805 patients with a recent nonmelanoma skin cancer (8).
In this issue of the Journal, Goodman et al. (9) report follow-up data for participants in the CARET study subsequent to the termination of the trial in 1996, discounting all of the cancer incidence and mortality events that had occurred before that date. Their findings complement the recently published findings of a similar follow-up study of the ATBC trial (10). Results of these two studies provide strong and consistent evidence that high-dose -carotene supplementation increases the incidence of lung cancer and the incidence of all-cause mortality in smokers. During the 6-year post-intervention follow-up of the CARET study, lung cancer incidence was elevated by 12% (95% confidence interval [CI] = 3% to 31%) among subjects who received the
-carotene/retinol supplements compared with subjects who received placebo, with most of the excess incidence occurring within the first 3 years after participants stopped receiving the
-carotene supplements, as indicated in the authors graphical analysis of relative risks over time. In the ATBC trial, the excess lung cancer incidence in the first 3 years following study termination was 17% (95% CI = 2% to 39%), and there was no excess risk in the subsequent 3 years. Although neither of these results was statistically significant, in both cases the results are an internal confirmation of the earlier published results from the active intervention phases of the trials, and the collective results provide strong evidence of the adverse effect of
-carotene supplement use on lung cancer incidence in smokers. Moreover, results of these two follow-up studies provide similar confirmation of the adverse effects of
-carotene on overall mortality. In the CARET study, the observed increase in overall mortality was 8% (95% CI = 3% to 17%), whereas the ATBC study reported overall mortality increases of 11% (95% CI = 3% to 21%) in the 3 years immediately following termination of the trial, 7% (95% CI = 1% to 15%) in the subsequent 3 years, and 1% (95% CI = 8% to 10%) in the subsequent 2 years, indicating a diminishing impact of the intervention after a period of 34 years, similar to that observed for lung cancer incidence.
Goodman et al. (9) have further analyzed their results and have drawn some conclusions about other hypotheses. For example, they suggested that the adverse effects of -carotene are restricted primarily to females and to former smokers. These conclusions must be considered speculative because they were based on excess relative risks in the post-intervention phase that appeared to be restricted to the female participants (relative risks of lung cancer for females versus males: 1.33 versus 1.08; relative risks of all-cause mortality for females versus males: 1.37 versus 1.00) and on their graphic representation of the temporal trends in relative risks, which showed no apparent decline among women over the entire post-intervention phase of the trial. However, these conclusions seem to be contradicted by two observations: during the intervention phase of CARET, males had excess risks of both lung cancer and all-cause mortality, and in the ATBC trial, a study that was restricted to male participants, strong effects for both outcomes were observed. Also, the conclusion by Goodman et al. that excess cardiovascular disease mortality is restricted to former smokers and women seems highly speculative because of the relatively small numbers of outcomes and the fact that compared with participants who received placebo, participants who received
-carotene had increased cardiovascular mortality in both the intervention and follow-up periods of the ATBC trial, a trial restricted to males who were current smokers at the outset of the study.
Chemoprevention researchers have learned much about the possible mechanisms of action of -carotene since the alarming results of the ATBC and CARET studies were published almost a decade ago. Indeed, many of the recent investigations into the biologically plausible mechanisms underlying these findings have been recently summarized (1113). The most widely accepted hypothesis is that the free radical-rich environment produced by chemicals in cigarette smoke and the resultant inflammatory response in the lung combine to induce oxidation of
-carotene, resulting in a proxidant effect (16). One of the most notable recent observations related to the
-carotene and smoke interaction comes from a cigarette smoke inhalation study in ferrets, an animal model that is similar to the situation in humans with respect to
-carotene absorption, accumulation of
-carotene in lung tissue, formation of oxidative metabolites, and pathologic response to cigarette smoke. That study showed that high-dose
-carotene (equivalent to the 30 mg of carotene per day used in the CARET) and smoke exposure (which produced urinary cotinine levels in animals that were similar to the urinary cotinine levels found in humans who smoke 1.5 packs of cigarettes per day) led to squamous metaplasia, an early precancerous lesion, in the lung (12). In addition, high dose
-carotene given to the smoke-exposed animals gave rise to a number of transient oxidative metabolites that stimulated the activity of the predominant cytochrome P450 enzyme in the lung, CYP1A1, resulting in the destruction of retinoic acid, diminished retinoid signaling (accompanied by increased expression of c-Fos, c-Jun, reduced expression of retinoic acid receptor-
[RAR-
]], and 3.7- and 1.8-fold increases in the levels of cellular proliferation marker PCNA in
-carotenesupplemented ferrets that were and were not exposed to smoke, respectively (12). These oxidative metabolites of
-carotene also facilitated the binding of cigarette smoke-derived carcinogens to DNA. Importantly, in this animal model, low physiologic doses of
-carotene (equivalent to the 6 mg of
-carotene per day attainable from a human diet high in fruits and vegetables) provided mild protection against cigarette smoke-induced squamous metaplasia.
These findings suggest that the adverse effects of high-dose -carotene on lung cancer incidence and overall mortality observed in the CARET and ATBC trials may be related to the pharmacologic doses of
-carotene used and the resultant supra-physiologic serum concentrations of
-carotene. This explanation is consistent with the apparent protective effect of
-carotene on lung cancer incidence and mortality reported in observational epidemiologic studies (24), as well as in the recently reported 10-year post-intervention follow-up of the General Population Nutrition Intervention Trial of the combination of 30 mg
-tocopherol, 15 mg
-carotene, and 50 µg selenium in a poorly nourished Chinese population, which showed continued protection against total and cancer mortality (14), and the null results from the PHS. Indeed, we agree with Forman and Altman (15), who noted that "the effect on disease with long latency periods of pharmacological doses of specific micronutrients over a few years in middle-aged adults is a different scenario from physiological doses of the same micronutrients provided as part of a balanced diet on a lifelong basis, starting in childhood". We also agree with Mayne et al. (16), who noted that "interventions aimed at restoring levels of a given nutrient in populations at nutritional risk... may be more effective than interventions that emphasize populations with adequate nutrient status and supplementing to supra-adequacy" (16).
The accepted paradigm for mounting chemoprevention trials has evolved since the CARET and ATBC trials were initiated. The current thinking is that phase I and II trials should be conducted prior to the onset of the larger, randomized, longer-duration phase III trials. In addition, a critical component of these preliminary trials is thought to be knowledge about the molecular mechanisms underlying the effect of treatment, which are evaluated through correlative studies of surrogate endpoint biomarkers, assuming that valid biomarkers in the dominant pathway mediating treatment effect are used, an assumption that is difficult to verify (17). However, it is unclear whether these steps would have predicted the adverse outcomes of the CARET and ATBC trials.
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