CHEMOPREVENTION OF LUNG CANCER: The Rise and Demise of Beta-Carotene

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1 Annu. Rev. Public Health :73 99 Copyright c 1998 by Annual Reviews. All rights reserved CHEMOPREVENTION OF LUNG CANCER: The Rise and Demise of Beta-Carotene Gilbert S. Omenn Former address: School of Public Health & Community Medicine, University of Washington, Seattle, Washington and Fred Hutchinson Cancer Research Center, Seattle, Washington ; Current address: University of Michigan, Ann Arbor, Michigan ; gomenn@umich.edu KEY WORDS: fruits/vegetables, carcinogenesis, vitamin-supplements, antioxidants, vitamin A ABSTRACT Beta-carotene and retinoids were the most promising agents against common cancers when the National Cancer Institute mounted a substantial program of population-based trials in the early 1980s. Both major lung cancer chemoprevention trials not only showed no benefit, but had significant increases in lung cancer incidence and in cardiovascular and total mortality. A new generation of laboratory research has been stimulated. Rational public health recommendations at this time include: 1. Five-A-Day servings of fruits and vegetables, a doubling of current mean intake; 2. systematic investigation of the covariates of extremes of fruit and vegetable intake; 3. discouragement of beta-carotene supplement use, due to adverse effects in smokers and no evidence of benefit in non-smokers; 4. multilevel research to develop and evaluate candidate chemoprevention agents to prevent lung and other common cancers; and 5. continued priority for smoking prevention, smoking cessation, and avoidance of known carcinogens in the environment. INTRODUCTION In 1981 Peto et al published a review in Nature (92) that brought together sufficient evidence of the potential cancer-preventing benefits of beta-carotene (and retinol) to stimulate direct tests of the hypothesis through randomized /98/ $

2 74 OMENN trials in human populations. The timing was propitious, as the National Cancer Institute (NCI) had begun to focus on prevention of cancers, to complement the basic biology, early detection, and treatment research programs supported by NCI. In the early 1980s, a portfolio of small and large randomized prevention trials was created, with beta-carotene the flagship agent to be investigated and with multiple studies of retinoids as well. Doll & Peto (20) also published a now-classic analysis of the preventable causes of cancer, deducing that about 30% of cancers were attributable to cigarette smoking, 35% to multiple other categories of exposures (infectious agents, alcohol, environmental pollution, occupational, geophysical), and the rest, very roughly 35%, to unspecified factors in diets. The search was on for explanations both positive and adverse for the striking differences among migrant populations and other ecologic correlations attributed to diet factors. As noted (92), the possibility of discovering anticancer substances that can be prescribed rather than carcinogens that must be proscribed is attractive, for more people may be willing to accept prescription than proscription. Particularly attractive would be agents that inhibit the late stages of the multistage carcinogenic process, since the benefits would be observable within, perhaps, 5 10 years, rather after a year latent period if the mechanism depended upon interference with initiation of carcinogenesis. Among the cancers, lung cancer emerged during this half-century as the leading cancer cause of death, accounting currently for 29% of cancer deaths and 6% of all deaths. Lung cancer has been the leading cause of cancer deaths in US men since the 1950s, and in US women since 1987 (118, 124). Indeed, many more women die of lung cancer than of breast cancer (22). Worldwide, lung cancer is the most common cause of cancer-related deaths, with an estimated 900,000 new cases and 785,000 deaths per year as of 1985 (87, 94). In its goals for the year 2000, the National Cancer Institute placed highest emphasis for lung cancer on prevention. Unlike many other common cancers, we do know the causes of the vast majority of lung cancers. Cigarette smoking accounts for about 90% of cases in men and about 80% of cases in women (101), with the rest due to various combinations of environmental tobacco smoke (second-hand smoke); occupational exposures to asbestos, arsenic, chloromethyl ethers, chromium-vi, and nickel; residential and occupational exposures to radon; and probably exposures to carcinogenic air pollutants. Risk factors include genetic factors (21, 80) and dietary factors (80, 124). Meanwhile, despite intensive efforts at early diagnosis and aggressive therapy, most lung cancers are not and cannot presently be detected until disease is advanced; five-year survival after diagnosis is still less than 15% (115). Thus, the need for primary prevention of lung cancer is paramount. Approaches can include both avoidance or reduction of these known causative

3 CHEMOPREVENTION OF LUNG CANCER 75 exposures and development of interventions for healthy individuals that modify the impact of risk factors and previous exposures in the carcinogenic process. EVIDENCE THAT JUSTIFIED TESTING BETA-CAROTENE AND VITAMIN A IN CHEMOPREVENTION TRIALS In 1981, there was already a substantial epidemiologic literature associating high fruits and vegetables intake, estimated beta-carotene intake in the diet, and blood levels of beta-carotene with lower incidence of epithelial cancers, particularly lung cancer. Five prospective and 15 retrospective case-control, questionnaire-based studies of populations in eight different countries were summarized by Peto et al (92). Some studies were opportunistic reanalyses of dietary assessments focusing on foods that accounted for the majority of the beta-carotene in that population: carrots in North America, yellow-green vegetables in Japan, red palm oil in West Africa and Brazil, and dark green leafy vegetables among Singapore Chinese. A relative risk was typically observed when comparing low versus high quartiles or quintiles of beta-carotene intake groups (roughly assessed by dietary questionnaires) for cancer risks (lung, stomach, colorectal, other). This epidemiologic association has been confirmed many times over in subsequent years and in the prevention trials to be highlighted in this review (see below; 2, 85). A dozen years later, 11 of 15 prospective and 10 of 17 retrospective epidemiologic studies had found statistically significant associations between low dietary or serum beta-carotene and increased risk of lung cancer (111). As Peto et al (92) had noted, it is most unlikely that this [inverse cancer risk] association will disappear entirely with future observational studies [but] the inverse association may be an artefact, due merely to association of beta-carotene ingestion with some truly protective dietary habit(s) or component(s) or avoidance of some truly harmful habits or components (92). The direct test, of course, of hypothesis-generating consistent associations arising from observational studies is the randomized population-based trial. By 1979, there were 324 research papers on retinol, other retinoids, betacarotene and other carotenoids cited in the International Cancer Research Data Bank (10). Animal experiments, beginning with Saffiotti et al in 1967 (99), had shown marked reductions in tumor yield and tumor mass, along with longer latency, when animals were treated with various retinoids before, during, and most importantly after exposure to initiating and/or promoting agents. Cultured cells transformed by viruses, chemicals, ionizing radiation, or tumor promoters underwent reversal of malignant changes upon exposure to retinoids.

4 76 OMENN Partly due to its very poor solubility in aqueous solution, beta-carotene had been studied much less than the retinoids in animals or in cell culture. Nevertheless, during the 1980s plenty of experimental evidence accumulated in favor of cancer prevention effects of beta-carotene in the diet or administered by injection or topical application (50, 53, 54); furthermore, combinations of beta-carotene with dietary vitamin A, 4-hydroxyretinamide (4HPR), oltipraz, or difluoromethylornithine (DFMO) all indicated additive chemopreventive benefits of beta-carotene. In NCI preclinical studies, beta-carotene and other carotenoids have inhibited tumorigenesis in head/neck, mammary, colon, bladder, and skin models in vivo and in mammary organ cultures in vitro (50). Deciding whether beta-carotene or retinol/retinoids should be the primary candidate for prevention trials and what doses should be used was speculative. The animal and cell culture studies, especially of natural and synthetic retinoids, provided strong support for a pharmacologic effect, not just overcoming a deficiency state, and for action late in the carcinogenic process. Testing beta-carotene was favored, in order to avoid the risk of hypervitaminosis A among participants who might be particularly susceptible or who might overdose themselves; no toxic effects of beta-carotene were expected, even at very high doses [dermatologists were comfortable with 180 mg beta-carotene/day for control of a photosensitive disorder called erythropoietic protoporphyria (65)]. On the other hand, retinol administration was recommended for really high-risk study populations, because of the greater evidence of its efficacy in animal studies. Besides questions about the direct effects of beta-carotene and the underlying mechanisms, there was the paradox in developed countries that beta-carotene contributes rather little to serum retinol levels, owing to inefficient cleavage and decreasing absorption rates when there are adequate body stores and intakes of preformed vitamin A, as well as homeostatic control of circulating retinol concentrations. Soon some intermediate endpoint human studies were published. The combination of beta-carotene and vitamin A reversibly reduced percent of oral mucosa cells with micronuclei among betel nut chewers (108); uncontrolled administration of etretinate (synthetic retinoid) to smokers in France was reported to be associated with improved histologic features on before-and-after bronchoscopic biopsies (72). CHEMISTRY AND PHARMACOLOGY OF BETA-CAROTENE AND VITAMIN A The term vitamin A is applied to two different families of dietary factors: (a) preformed vitamin A, chiefly retinyl esters, plus retinol and retinal; (b) provitamin

5 CHEMOPREVENTION OF LUNG CANCER 77 A, chiefly beta-carotene, plus numerous other carotenoids, some of which can be converted enzymatically in the intestine to retinal and retinol. Beta-carotene is a naturally occurring carotenoid pigment widely distributed in vegetables and fruits (50, 53, 54). Of 700 known naturally occurring carotenoids, 53 serve as precursors to vitamin A. If the aldehyde retinal (C20, H28, O) is written R=0, then retinol is H-R-OH, retinoic acid H-R-OOH, and all-trans-beta-carotene R=R. Thus, beta-carotene and most other carotenoids are based on a 40-carbon skeleton, with 3 to 15 conjugated polyene C=C double bonds. The full chemical name for beta-carotene is (all-e)-1,1 -(3,7,12,16-tetramethyl-1,3,5,7,9,11,13,15,17-octadecanonaene, 1,18-diyl)bis(2,6,6-trimethylcyclohexene). Carotenoids are isoprenoid compounds biosynthesized by tail-to-tail linkage of two C20 geranylgeranyl diphosphate molecules, which were formed by tail-to-tail linkage of four isoprene molecules (11). The system of alternating double and single C-C bonds forms the central part of the molecule, with pi-electrons delocalized over the length of the polyene chain generating the distinctive molecular shape, chemical reactivity, and light-absorbing properties (11). Light absorption occurs in the low-energy, visible range of the spectrum, giving fruits and plants their brilliant yellow, orange, red, and green colors. Vitamin A (retinol ingested or derived by cleavage of retinyl esters or betacarotene) is absorbed from the intestine, esterified and stored in the liver, and released in controlled amounts that maintain plasma retinol levels in a narrow range mostly attached to the retinol-binding protein (RBP) produced in the liver. In severe vitamin A deficiency, RBP accumulates in the liver; in severe vitamin A excess, the binding capacity will be exceeded. Oral contraceptive users have elevated blood retinol levels, reflecting elevations of RBP (and other plasma proteins). Wachenroder is credited with coining the name carotene for the hydrocarbon pigment he crystallized from carrot roots in Berzelius in 1837 named the more polar yellow pigments extracted from autumn leaves xanthophylls, and Tswett in 1911 separated carotenoids chromatographically. The enzymes and genes involved in carotenoid biosynthesis in several bacterial and plant species have now been identified (see 115). The seven predominant carotenoids in human circulating blood are beta-carotene, lycopene, lutein, alpha-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, and zeaxanthin. Absorption is highly variable among individuals after either single or multiple doses, and is enhanced by dietary fat and influenced by overall nutritional status. About 15% of betacarotene is cleaved into retinal in the intestine in poorly nourished humans; that percentage decreases as the amount of beta-carotene in the diet increases, so that even massive doses of beta-carotene do not yield enough intestinederived retinol to cause hypervitaminosis A. About 25 75% of beta-carotene

6 78 OMENN just goes through the intestine and out in the feces, but the remainder is absorbed unchanged, circulates throughout the body, and is stored in adipose tissue, owing to its fat-solubility, giving fat its yellow color. Elimination occurs slowly through the fecal route. Different carotenoids are concentrated in different organs: lycopene and beta-carotene in the prostate, and lutein and zeaxanthin in the macula of the eye, for examples. The absorption and distribution of beta-carotene are influenced by retinol intake, which suppresses conversion to vitamin A and, therefore, increases circulating levels of beta-carotene in response to a given dose of beta-carotene. Rodents have low serum carotenoid levels (ng/ml range, about 1/1000th of human levels) that are not related to dietary intake, due to very active 15,15 - dioxygenase cleavage to retinal. In the ferret model and probably in humans, eccentric cleavage of beta-carotene and certain other carotenoids can form retinoic acid (78). There is controversy at present about whether the fat substitute called olestra (a long-chain fatty-acid polyester of sucrose) could sequester enough betacarotene and other phytochemicals in the intestine or fail to provide the needed fat-facilitated absorption to significantly reduce serum levels and whether such reductions would have adverse health consequences. Experimental data compiled on the effects of olestra on the availability of 29 compounds, mainly nutrients and oral medications, showed that olestra affects only molecules having octanol-water partition coefficients greater than about 7.5 (very poorly soluble in water). Potential reductions in the average effective beta-carotene intake were calculated for free-living individuals eating olestra-containing snack foods. For extreme scenarios, among individuals with the lowest 10% of carotenoid intakes, the estimated reduction was 6%; for heavy snack eaters (top 10%), the estimate was 9.5%. Similar effects can occur with other dietary factors (18). A large-scale postmarketing study is being conducted to measure any such effects (M Thornquist, unpublished data). Mechanisms of Action Beta-carotene is most associated with a potent singlet oxygen quenching capacity, thought to be highly relevant to prevention of lipid peroxidation and inactivation of various metabolically generated free-radical species in cancer cells, other cells, and circulating low-density lipoproteins (11, 13, 53). However, many of its effects cannot be explained by singlet oxygen quenching. Both electrontransfer physical effects (catalytic formation and then rotational/vibrational dissipation of the triplet excited state of the carotenoid) and chemical reactions (consuming the beta-carotene or oxidizing the beta-carotene) are involved in inactivating peroxy and alkoxy radical species, and superoxide anion. Radical addition to carotenoids from lipid peroxyl radicals can be induced by the

7 CHEMOPREVENTION OF LUNG CANCER 79 effect on lipid membranes of singlet oxygen, nitrogen dioxide, or thiyl radical (derived from the interaction of NO2 with glutathione) (23, 29). Carotenoids differ in their propensity to react through these various pathways with various radicals and in different in vitro test systems. For example, beta-carotene forms an adduct with singlet oxygen, whereas lycopene undergoes electron transfer. The xanthophylls astaxanthin and canthaxanthine are better than betacarotene at inhibiting lipid hydroperoxide formation in homogeneous milieu using methyl-linoleate and azo compounds; these keto-group-containing xanthophylls behave as molecular rivets across bilayer membranes (97). The complex chemistry of carotenoid action in vivo is under investigation. Beta-carotene and other carotenoids that are vitamin A precursors may exert their effects through cleavage to retinol, which inhibits proliferation and induces differentiation of epithelial cells through direct binding to RAR and RXR nuclear receptors and subsequent modification of gene activity (91). In addition, carotenoids themselves have been reported to modulate cytochrome P450 metabolism (beta-carotene, cryptoxanthin, lutein) (100); inhibit arachidonic acid metabolism (beta-carotene (34); act as antioxidants and free radical/reactive species scavengers (astaxanthin, canthaxanthine, alpha-carotene, beta-carotene, crocetin, cryptoxanthin, lutein, lycopene, zeaxanthin) (23, 110); modulate immune functions (astaxanthin, canthaxanthine, beta-carotene) (47); induce differentiation and/or gap junction intercellular communication (astaxanthin, canthaxanthine, alpha-carotene, beta carotene, lutein, lycopene) (123); inhibit chromosome instability and chromosome damage (canthaxanthine, betacarotene) (55); and inhibit biochemical changes associated with proliferation, including N-myc expression (alpha-carotene, beta-carotene) (26), ornithine decarboxylase (ODC) activity (beta-carotene) (93), and adenylate and guanylate cyclase activity (beta-carotene) (37). Beta-carotene may also influence apoptosis, which leads to cell death instead of cell proliferation; bcl-2 gene activity decreases reactive oxygen species (48, 52), has an anti-apoptosis effect, and enhances cell proliferation (41). Alterations in oxidant/antioxidant balance (29) might be mediated through such mechanisms. The apparent lack of toxicity in high-dose clinical use against erythropoietic protoporphyria (65), the impressive epidemiologic association with lower cancer incidence (above), and these biological properties combined to justify cancer prevention trials in humans. THE NCI PORTFOLIO OF BETA-CAROTENE AND RETINOID CHEMOPREVENTION TRIALS By the mid-1980s, the NCI launched large-scale human trials of beta-carotene alone against skin cancer (32), colonic polyps (33), and total cancers (40), and

8 80 OMENN of beta-carotene and/or vitamin E (2) and beta-carotene plus retinol (85) against lung cancer. Shorter-term trials using sputum for presumed intermediate endpoint were conducted as well. In Europe, van Poppel et al (113) reported that 20 mg beta-carotene/day in 114 male smokers led to a 27% decrease (CI 9 41%) in micronuclei in sputum. Heimburger et al (39) had reported an improvement in a cytological score for sputum atypia in 73 male smokers with metaplasia on bronchial biopsy, using 10 mg folate plus 500 µg B12; however, the analysis has been challenged (59). Studies with oral micronuclei (108) and bronchial biopsies (72) were noted above. The major trial with sputum endpoints, using the CARET intervention of beta-carotene plus retinol (see below) in 755 males with extensive asbestos manufacturing exposure in Tyler, Texas, found no benefit and an odds ratio of 1.24 (CI ) (59). Another type of trial population is comprised of patients who are survivors of lung or head and neck cancer at risk for recurrences and second primaries. Pastorino et al (90) used 300,000 IU vitamin A in 307 patients who had been treated curatively for stage I non-small-cell lung cancer and found a longer time to second cancer (p = 0.045) in those in the vitamin A treatment arm of this secondary prevention trial. A much larger trial, Euroscan (19), testing the combination of 300,000 IU vitamin A plus 600 mg N-acetylcysteine in 2000 patients surviving lung, oral, and laryngeal cancers, is due to report soon. Finally, Mayne et al (69) are conducting a trial of 50 mg beta-carotene/day in 480 US patients with oral, pharyngeal, and laryngeal cancers, with reduction in second cancers the endpoint. The Alpha-Tocopherol/Beta-Carotene (ATBC) Trial in Finland Investigators at the National Public Health Institute at the University of Helsinki (led by O Heinonen & J Huutunen) and collaborators at the NCI in the United States (led by D Albanes & P Taylor) organized a primary prevention trial among 29,133 male smokers, age Their smoking histories averaged one pack/day for 36 years. The 2 2 factorial design evaluated 20 mg Roche betacarotene and/or 50 IU alpha-tocopherol (vitamin E) daily for 6.5 years. These doses represented a fivefold excess over the median dietary intake of alphatocopherol (10 mg) and a tenfold excess over the median intake of beta-carotene (1.7 mg) in this population (2, 3, 124). Follow-up was greatly facilitated by the national disease registry in Finland; 874 lung cancer cases and 3570 deaths were ascertained. The results (Table 1) were stunning: Vitamin E supplementation did not reduce the incidence of lung cancer [relative risk (RR) 0.98], and participants receiving beta-carotene alone or in combination with alpha-tocopherol had

9 CHEMOPREVENTION OF LUNG CANCER 81 Table 1 Relative risk comparisons Lung cancer Mortality Relative 95% Relative 95% Risk CI Risk CI ATBC CARET PHS Linxian Study significantly higher lung cancer incidence (RR 1.18; 95% CI ) and higher total mortality (RR 1.08, CI ) than participants receiving placebos. The Finnish study was well conducted, with excellent follow-up, full ascertainment of endpoints, and extensive review by an independent Data Monitoring Board and by an ad hoc group of investigators convened by the NCI before publication. The excess lung cancer incidence and excess mortality were not apparent in the initial 18 months; thereafter, the incidence curves in the study arms diverged and continued to diverge to generate the statistically significant differences. The impact of this crucial trial in the scientific community and in the media was muted, due to disbelief and continued reliance on the evidence, confirmed in the ATBC Study, of the inverse relationship between baseline beta-carotene serum levels and subsequent risk of lung cancer. However, the NCI portfolio approach generated striking, confirmatory evidence of both lack of benefit and adverse effects. Subsequent subgroup analyses (1) noted a higher point estimate of lung cancer risk in smokers of 20+ cigarettes/day (RR 1.25, CI ) than in light smokers (5 19 cigarettes/day) (RR 0.97, CI ), but these confidence intervals clearly overlap. These analyses also indicated associations with alcohol intake and with non-small-cell histology. The Beta-Carotene and Retinol Efficacy Trial (CARET) in the United States CARET is a multicenter, two-armed, double-masked randomized chemoprevention trial conducted in six study centers in the United States. The aim was to test whether oral administration of the combination of beta-carotene (30 mg/day) and retinyl palmitate (25,000 IU/day) can decrease the incidence of lung cancer in high-risk populations (109). The intervention was designed to combine the presumed antioxidant effects of beta-carotene and the tumorsuppressing, differentiation-enhancing molecular actions of vitamin A (see above). CARET successfully randomized 1845 participants in its

10 82 OMENN pilot phase (31, 88), plus 16,469 participants between 1989 and 1994; of the 18,314 total participants, 4060 are asbestos-exposed males, age 45 74, and 14,254 are smokers and former smokers (44% female), age at enrollment. CARET stands out as one of the first large trials to recruit women, long before Congressional and NIH policy mandates. The design was based on accumulation of 108,000 person-years by October 1997, with power to detect a 23% reduction in lung cancer incidence in the combined populations, as well as 27%, 49%, 32%, 35%, and 35% reductions in the smoker, female smoker, male smoker, asbestos-exposed, and former-smoker subgroups, respectively. CARET also had substantial power to detect possible chemopreventive effects on coronary heart disease plus sudden cardiac death, on cataract extraction rates, and on prostate cancer incidence. The pilot studies demonstrated successful recruitment from asbestos worker sources (88) and from health insurance sources (31) in the Seattle area, and the full accrual nationally was accomplished in timely fashion. Recruitment at the final study center in southern California was complicated by the great popularity of beta-carotene supplementation by the early 1990s, including addition of betacarotene to cereals. Many otherwise eligible and interested individuals declined to accept randomization that would put half of them into the placebo arm of the trial. Ironically, when the ATBC results were set to appear in April 1994 in the New England Journal of Medicine (2) and in the news media, the CARET PI sent individual letters to each CARET participant describing the ATBC results as important, surprising, and unexpected, encouraging them to continue as our partners in this research, but reminding them that they were both welcome to ask questions of the CARET research team and entitled to stop taking their supplements if they so chose. Of the 18,314 participants, 606 did choose to stop taking their capsules, 3.8% of all living, active participants. The accrual of endpoints was followed closely by the principal investigator and chief biostatistician of CARET and by the independent CARET Safety and Endpoints Monitoring Committee, especially after the ATBC results became known. All other CARET investigators were blinded to endpoints analyses, of course. The scheduled second interim analysis was moved forward a year, to late After extensive analyses and review, an extraordinary meeting of the CARET Steering Committee was convened on January 11, 1996, to reveal the findings and the recommendations of the CARET and NCI review groups and to consider appropriate action. There was clear evidence of no benefit, and substantial, accumulating evidence of persistently greater incidence of lung cancer and mortality in the active treatment group, beginning about 24 months after randomization. The Steering Committee considered the importance of drawing a correct conclusion, the significance of distinguishing between a null effect and an adverse effect, and the likelihood that no future study of

11 CHEMOPREVENTION OF LUNG CANCER 83 beta-carotene would be conducted. We were aware, as noted previously (85), that reversals of interim findings have occurred in the course of a single randomized trial (42, 44) and that opposite conclusions have been reached after additional trials of the same agent or class of agents (4, 43, 122). We also discussed the awkwardness of confronting such information without any warning for almost all of the Steering Committee, due to the double-masked nature of the protocol. The CARET evidence, combined with ATBC, was sufficiently compelling, and the obligation to protect the lives of CARET participants so clear, that the Steering Committee voted unanimously to terminate the active intervention immediately. Over the next seven days letters were sent to each participant with information about the results and instructions to stop taking the capsules and return them to the study centers; a manuscript was prepared and submitted for publication, as prearranged, to the New England Journal of Medicine, which waived its prohibition on prior publicity, because of the public health importance of the findings; investigators heading other trials using beta-carotene were informed; and the NCI Director and CARET PI Omenn held a joint press conference in Bethesda on January 18 to report the findings. Media coverage was intense. The findings were these (85): A total of 388 new cases of lung cancer were diagnosed during the 73,135 person-years of follow-up (mean 4.0 years). The active-treatment group had a relative risk (RR) of lung cancer of 1.28 (CI , p = 0.02), compared with the placebo group. These point estimates were greater than 1.1 for the smokers, asbestos-exposed, male, female, vanguard, and efficacy subcohorts; furthermore, the differences were becoming greater as the intervention progressed (see Figure 1). There were no statistically significant differences in the risks of other types of cancers. In the active-treatment group, the relative risk of death from any cause was 1.17 (CI ), of death from lung cancer 1.46 (CI ), and of death from cardiovascular disease 1.26 (CI ). Actually, these were unweighted analyses, since the Journal insisted that more sophisticated analyses should be withheld and published in a specialty journal. Those analyses, including subgroups, weighting of endpoints over the first 24 months (full effect of benefit or risk reached only after 2 years on treatment), and sensitivity analyses, were published in the Journal of the National Cancer Institute (86). With the prespecified primary analytic method, using a stratified logrank test to estimate relative risks with Cox regression models, the RR were 1.36 (CI , p = 0.01) for weighted lung cancer incidence and 1.59 (CI , p = 0.01) for weighted lung cancer mortality. All subgroups, except former smokers, had a point estimate of RR of 1.10 or greater for lung cancer incidence. As in further analyses from ATBC published in the same

12 84 OMENN Figure 1 Kaplan-Meier curves of the cumulative incidence of lung cancer among participants receiving active vitamins and those receiving placebo. Data are shown only through 5.5 years of follow-up because of the small number of participants beyond that time. issue (1), there was an association of the excess lung cancer incidence between treatment groups with the highest quartile (rather modest levels) of alcohol intake, but no association with baseline serum beta-carotene concentrations. The point estimates in CARET are greater than in ATBC, primarily reflecting more extreme results in the asbestos-exposed cohort of CARET. In addition, CARET used vitamin A; we could not distinguish effects of beta-carotene from those of vitamin A, since the two agents were administered in combination. Retinol has been associated with increased risk of cancer in some studies at various sites (67); epidemiologic or experimental evidence of prostate and pancreas increased risk was explicitly cited in the CARET informed consent documents. Both the ATBC Study population and the CARET population are being followed for posttreatment accrual of additional endpoints. Of particular interest is the status of the former-smoker subgroup (see below). In addition, CARET is conducting multiple ancillary studies that may shed light on the mechanisms of the adverse effect. Other Trials TYLER ASBESTOS-MANUFACTURING COHORT McLarty (70) randomized 755 asbestos workers mostly identified in the follow-up of workers at an asbestosmanufacturing plant in Tyler, Texas, to receive either 50 mg beta-carotene and 25,000 IU retinol/day or placebos to see if the combination could reduce

13 CHEMOPREVENTION OF LUNG CANCER 85 the prevalence of atypical cells in sputum. The intervention was based directly on the CARET rationale and choice of agents. The beta-carotene was obtained from BASF, while CARET was awaiting provision of beta-carotene from Hoffman La Roche; the 50 mg and 30 mg dosages, respectively, from these two sources are thought to be equivalent. After a mean intervention period of 58 months, there was no difference in the two groups by criteria for sputum atypia. PHYSICIANS HEALTH STUDY This long-term trial was organized to test the effect of aspirin on cardiovascular disease incidence (106). Beta-carotene was added in a 2 2 design, using 50 mg BASF beta-carotene on alternate days. Using an efficient design depending upon physicians presumed to be knowledgeable for self-monitoring and self-reports, investigators at the Harvard Medical School followed the 22,071 male physicians for a mean of 12.5 years of intervention through the end of In this healthy population, with 50% never-smokers and only 11% current smokers, only 170 lung cancers were accumulated over the long follow-up period. The relative risks were 1.02 (CI ) for overall mortality, 0.98 (CI ) for all malignant neoplasms, and 0.93 for lung cancer. The authors concluded that the trial yielded a firm no benefit, with no evidence of harm, either. Earlier they had been hopeful that prostate cancer or other endpoints might be reduced in incidence with extended follow-up, but the long follow-up generated considerable weight for the conclusion of no benefit. LINXIAN A pair of notable trials was conducted from 1986 to 1991 in Linxian County, China. This site was chosen because of very high incidence of esophageal and gastric cardia cancers, the stability and accessibility of the population, and the presence of a team of able collaborators in China. The population was characterized as having low serum concentrations of multiple micronutrients, including beta-carotene, retinol, riboflavin, vitamin C, and vitamin E. The larger of the trials (7) involved 29,584 men and women from the general population, age They were randomized into a complex factorial design using different nutrient combinations. The primary endpoint was incidence of esophageal and gastric cancers. The combinations were (A) retinol plus zinc, (B) riboflavin plus niacin, (C) vitamin C plus molybdenum, and (D) 15 mg beta-carotene, 50 µg selenium, and 30 mg alpha-tocopherol (vitamin E). Notably lacking in this study was folic acid (see below). At the end of five years, none of the combinations had produced a significant reduction in the primary endpoint of esophageal and gastric cardia cancer incidence. However, the groups receiving (D) fared best, with RR 0.87 (CI ) for total cancer deaths, 0.91 (CI ) for total deaths, 0.96 (CI ) for esophageal cancer deaths, and 0.79 (CI ) for gastric cancer deaths. A tantalizing finding is the

14 86 OMENN note that the 31 lung cancer deaths in Linxian were distributed among the eight treatment groups so as to yield an estimated RR for the antioxidant combination (D) of 0.55 (CI ) (8). The second trial, in 3318 Linxian residents at high risk for esophageal cancer because they were already diagnosed with esophageal dysplasia, utilized a single supplement containing 26 vitamins and minerals (including folic acid, B6, and B12), plus a capsule with 15 mg beta-carotene (58). After six years, the corresponding relative risks were 0.92 (CI ) for esophageal-plusgastric cardia deaths, 0.84 (CI ) for esophageal cancer deaths, 0.93 (CI ) for total mortality, and 0.96 (CI ) for cancer mortality in the supplemented group. Stomach cancer mortality, which was statistically significantly reduced in the Linxian general population (above), was higher, with RR 1.18 (CI ). None of these many results in the Dysplasia Trial was statistically significant. In sum, no marked benefit could be demonstrated during the 5 6 years of intervention, but several point estimates of relative risk pointed to potential benefit from vitamin supplementation. Secondary analyses from Linxian include reports about stroke, blood pressure, and cataract endpoints (62, 63, 102). These Linxian Trials are very complex in design. Although a clear alternative hypothesis would have been correction of deficiency status for the various vitamins and minerals, the dosages tested in (D) were definitely in the pharmacologic range. Analysis of the results requires the simplifying assumption that the risk factors are additive on a log scale and have no interactions, which surely is violated in the groups receiving (A) and (D), since administration of retinol suppresses cleavage and conversion of beta-carotene to vitamin A, for example. It is frustrating that there are no consistent findings between the Dysplasia and General Population studies. Nevertheless, it will always be difficult to relate the findings in this nutritionally deficient population to the findings in western populations (66), let alone the specific findings with regard to lung cancer. INTERPRETATION OF THE RESULTS OF THE MAIN LUNG CANCER CHEMOPREVENTION TRIALS If the ATBC Study and CARET had found lower incidence of lung cancer, as hypothesized from the inverse associations of fruits and vegetables and of betacarotene intake and blood levels with lung cancer risk, the interpretation would have focused on the appropriate dose, the comparative virtues of supplementation versus greater dietary intake, and the associated benefits for other endpoints. If the studies had shown no effect (null result), the interpretation would be wide open, with the focus on possible inadequate dose for pharmacologic effect (compared with dosages in rodents), on possible inadequate duration of

15 CHEMOPREVENTION OF LUNG CANCER 87 intervention, on need to start earlier in life, or on need for combinations of betacarotene with other compounds in fruits and vegetables. In striking contrast, the finding and the confirmation of the finding of major adverse effects more lung cancers, more cardiovascular deaths, and higher overall mortality have forced scientists, regulators, and health educators to re-examine our scientific knowledge about the effects of beta-carotene and to re-think our recommendations for diet and for supplements. Particularly influential in assessments of these trials is the clear pattern of increase in adverse effect with longer duration of supplementation (Figure 1), with no differences in the initial months. A pooled analysis (86) of the unweighted results of ATBC plus CARET plus PHS yielded a RR of 1.16 (CI ) for lung cancer incidence and RR 1.07 (CI ) for all-cause mortality. Is Beta-Carotene Really a Good Antioxidant? Many recent studies have cast doubt on the effectiveness of beta-carotene, especially in vivo, as an antioxidant. The powerful logic that beta-carotene should help prevent coronary heart disease by protecting against oxidation of lowdensity lipoproteins in the LDL particles, which actually carry beta-carotene in the lipid phase of plasma, has been torpedoed by direct study: Administration of beta-carotene does not reduce oxidation of LDL in vivo (27, 95, 96). Similarly, it was thought that antioxidant actions might account for reduced micronuclei counts in sputum from heavy smokers given 20 mg beta-carotene/day for 14 weeks in a randomized trial (71), but direct measurement of oxidative damage, using 8-oxo-7,8-hydro-2 -deoxyguanosine as a marker in urine, failed to show any antioxidant activity in vivo (112). Beta-carotene has long been described as an unusual antioxidant (13). Experts in redox chemistry have begun to point out that many chemicals may be either prooxidants or antioxidants, depending upon the other chemicals present and the characteristics of the microenvironment. Given the adverse effects demonstrated in ATBC and CARET, vitamin supplement enthusiasts and investigators have turned their attention to vitamin E, vitamin C, coenzyme Q, other carotenoids, and other presumed antioxidants (64). Such other agents might actually protect against oxidation of beta-carotene (see below), perhaps contributing to the apparent protective effect of higher fruits and vegetables intake seen in ATBC and CARET within the placebo and the active-treatment groups. Could Current Smoking Account for the Adverse Actions of Beta-Carotene? The results put the spotlight on current smokers in the ATBC Study and current smokers (with or without asbestos exposure) in CARET. Subgroup analyses (1, 86) indicate that the adverse effects of supplemental beta-carotene in both

16 88 OMENN trials might be restricted to persons who smoked one pack of cigarettes or more per day or who drank above-average intakes of alcohol. Conversely, since the low incidence of lung cancer in the Physicians Health Study (40) is surely explained by the low prevalence and low intensity of smoking in that population, the lack of adverse effect might be explained similarly. The gas phase of cigarette smoke is loaded with free radicals and is highly oxidative (16, 68, 104). Under conditions of high oxidative stress and phagocytemediated inflammatory responses, beta-carotene and other carotenoids may be oxidized to epoxides and other reactive derivatives, which then act as prooxidants (36, 51, 121) and as chemical messengers that stimulate cellular proliferation, especially in carcinogen-altered epithelial cells (45, 73). Exposure of plasma to cigarette smoke led to oxidation and destruction of carotenoids, as well as alpha-tocopherol, but not plasma lipids (35, 45). Oxidized betacarotene increases the formation of benzo[a]pyrene-dna adducts when B[a]P is incubated in vitro with rat microsomes, while beta-carotene itself decreased such adduct concentrations (WA Pryor, personal communication). Since autooxidation of beta-carotene in vitro is dose dependent, Mayne et al emphasized that mean serum levels of beta-carotene in the supplemented group in the Physicians Health Study (1200 ng/ml) were lower than those in CARET (2100 ng/ml) and ATBC (3000 ng/ml) (68). However, all of these levels represent a very large multiple from baseline ( ng/ml), and there was no evidence in ATBC or in CARET that the excess lung cancer cases occurred disproportionately among participants in the upper tertile of serum beta-carotene values within the active treatment arm. Asbestos exposure might also involve oxidative mechanisms (45). Iron, a catalyst for oxidation, is a constituent of asbestos fibers. Inflammatory cells recovered by bronchoalveolar lavage from nonsmokers with asbestosis spontaneously release increased amounts of superoxide anion and hydrogen peroxide, compared with normal individuals (98). While monitoring longitudinally potential impacts of the intervention on pulmonary function, CARET investigators have reported that higher prerandomization baseline serum beta-carotene values are associated with better pulmonary function (FEV-1, FVC) (17). Mayne et al (68) proposed an analysis of the effects of the CARET intervention in those with asbestosis versus those with asbestos exposure but no manifestations of asbestosis. Could Beta-Carotene, Therefore, Be Protective in Nonsmokers and Former Smokers? The point estimates in CARET subgroup analyses (83) raise the tantalizing possibility that former smokers might actually benefit. Among the CARET heavy smoker population, those who were former smokers at baseline had a relative

17 CHEMOPREVENTION OF LUNG CANCER 89 risk of 0.80 for lung cancer in the active treatment arm, compared with former smokers in the placebo arm. It would be highly reinforcing to stop-smoking programs if we could recommend beta-carotene or beta-carotene plus retinyl palmitate (the CARET combination) to former smokers. However, the 95% confidence interval on that point estimate is , making any interpretation of benefit quite uncertain. More devastating are the unfavorable relative risks for overall mortality (RR 1.06) and cardiovascular mortality (RR 1.28) in the former heavy smoker group. Furthermore, no support for a protective effect was found in the asbestos-exposed former-smoker subpopulation of CARET (RR 1.47, CI ) (86). Might Beta-Carotene Be Effective Only in Individuals with Very Low Baseline Beta-Carotene Levels? In CARET and in numerous case-control studies, those in the lowest quintile or quartile of (baseline) beta-carotene intake or serum level have the most risk, and most of the difference occurs in the move up to the second quintile or quartile. Maybe trials should be directed at those who are prescreened and shown to have low beta-carotene baseline values, once enough is learned about intraindividual variation. A dose of 1 2 mg might be appropriate, either through diet or supplements. At this point, however, there is no affirmative evidence from trials in support of this hypothesis, not even the Linxian trials (7, 58). What Else Does Beta-Carotene Do? Beta-carotene and other vitamins are chemicals that require toxicologic characterization just like other chemicals, especially when they are proposed for use in pharmacologic doses. Even at physiologic concentrations, we lack much knowledge about their potential diverse effects. In the section above on chemistry and pharmacology, several reports of potentially favorable effects are cited (23, 26, 29, 34, 37, 47, 55, 91, 93, 97, 100, 110, 123). The trials results have stimulated a new generation of investigations of the potential unfavorable effects of this agent, which should yield numerous publications in the next few years. Recent papers show dietary beta-carotene has increased papilloma formation and tumor yield per animal in DMBA-treated Sencar mice (14) and respiratory tract cancers in hamsters exposed to benzo[a]pyrene (119). One specific possibility is that beta-carotene supplementation in substantial doses somehow unbalances circulating or tissue levels of other antioxidants or carotenoids or other key compounds. It was reported that administration of 15 to 60 mg/day beta-carotene in young adult volunteers produced a striking decline in serum alpha-tocopherol levels (120), but at least four major studies since have refuted that finding (30, 70, 77), and the original authors have not been able to confirm their findings. Preliminary studies within CARET of levels of

18 90 OMENN multiple other carotenoids indicate no striking changes from the beta-carotene plus retinyl palmitate treatment; similarly reassuring results were found by Gaziano et al for alpha-tocopherol and ubiquinol, but there was a modest decline in lycopene noted (28). Is There Any Reason to Prefer Natural Beta-Carotene over Synthetic Beta-Carotene? Health food advocates have made many claims that beta-carotene from fruits and vegetables or from algae might be protective and safe, even if synthetic all-trans beta-carotene is not. There is no scientific basis for such claims. The beta-carotene in fruits and vegetables is indistinguishable chemically from the synthetic all-trans compound; both have traces of 9-cis and 13-cis isomers (28, 74). The algal extracts have an approximately 1:1 mixture of all-trans and cis isomers, including the 9-cis isomer that is thought to be the direct precursor of 9-cis-retinal and 9-cis-retinoic acid, the endogenous ligand for the vitamin A RXR/RAR receptors in the nucleus of cells (91). However, three independent feeding studies have shown that the 9-cis-beta-carotene is either not much absorbed or is mostly converted into the sterically more stable alltrans form upon absorption from the intestine (28, 46, 103). Therefore, there is no scientific basis to prefer beta-carotene purified from natural sources over the synthetic product. Interest continues to grow in the 9-cis compounds. 9-cis-beta-carotene can arise in tissues at 37 C from all-trans; 9-cis retinol dehydrogenase converts the 9-cis isomer to the highly active retinoid 9-cis-retinoic acid (K Swisshelm, J Paik, W Blaner, personal communication). If Not Beta-Carotene, What Else in Fruits and Vegetables Could Be Chemoprotective? There are numerous candidates. One major component generally neglected in cancer epidemiology is folic acid. Folic acid has emerged as a crucial supplement for prevention of neural tube birth defects (74); FDA has mandated fortification of wheat grains with 140 µg folic acid per 100 gm grains, beginning in January 1998 (24). Folic acid is a leading candidate for reduction of homocysteine levels and potential large reductions in coronary heart, cerebrovascular, and peripheral vascular disease endpoints (5, 9, 60, 74, 75, 84). Folic acid is abundant in many of the same fruits and vegetables that are rich in carotenoids (81). In fact, the correlations for folic acid, estimated from the four-day diet record data from the Women s Health Trial Feasibility Study in Minority Populations, are 0.50 with total vitamin A, 0.51 with retinol, 0.30 with beta-carotene, and 0.52 with alpha-tocopherol (A Kristal, personal communication). Folic acid, vitamin B12, methionine, and choline are intimately

19 CHEMOPREVENTION OF LUNG CANCER 91 interrelated in methyl group metabolism. Much basic cancer research has focused on DNA methylation, and hypomethylation has been associated particularly with colorectal carcinoma. A methyl-deficient diet (deficient in choline, methionine, and folic acid) produced imbalances in deoxynucleotide pools and hypomethylation of CCGG sites (15). Several case-control studies (81) have yielded impressive inverse associations with colon cancer or adenomatous polyp incidence. Early studies suggested a favorable influence on cervical cancer, but recent studies, including a trial with cervical carcinoma in situ at the Fred Hutchinson Cancer Research Center (J Chu, personal communication) were unable to demonstrate a benefit. Heimburger et al (39) conducted a randomized trial of 10 mg folate plus 500 µg B12 versus placebos in 73 men with a history of more than 20 years of cigarette smoking who had metaplasia in sputum cytology. Four months of treatment yielded significantly greater reduction of atypia in the supplemented group. The results were interpreted very cautiously and apparently have not been replicated. These doses produced fold increases in mean plasma folate, 3 4-fold increases in mean red blood cell folate, a 30% increase in plasma B12, with no changes in retinol, beta-carotene, vitamin C, or vitamin E levels. Interestingly, these investigators used hydroxocobalamin rather than the usual pharmacological form of B12, cyanobalamin, to scavenge possible excess cyanide compounds resulting from reaction of tissue cobalamins with cyanides in cigarette smoke and to avoid increasing the cyanocobalamin pool. Lycopene and other carotenoids are popular candidates as well. With the perspective of the beta-carotene trials, however, we should be cautious about assuming that such chemicals are safe in pharmacologic doses, let alone efficacious, just based on epidemiologic associations, which entail multiple comparisons. Another component of fruits and vegetables under development in the NCI program is D-glucaric acid (114). One of its derivatives is the potent betaglucuronidase inhibitor D-glucaro-1,4-lactone. The lactone increases detoxification of drugs, steroid hormones, chemical carcinogens, and tumor promoters by inhibiting beta-glucuronidase, thereby preventing hydrolysis and release of the active compounds from the inactive glucuronidated conjugates before excretion in the bile or urine. A positive correlation has been found between the serum beta-glucuronidase level and susceptibility to chemical carcinogenesis in various strains of mice and rats (114). Calcium glucarate effectively inhibited B[a]Pinduced transformation in a rat tracheal epithelial cell assay (105). Calcium glucarate has been shown to inhibit colon and mammary gland carcinogenesis in rodents, alone or in combination with retinoids (4-hydroxyphenylretinamide and 13-cis-retinoic acid) or with vitamin D3 (116). A promising rationale has been developed for considering this calcium D-glucarate as a chemopreventive