A Randomized Trial

Shumin M. Zhang, MD, ScD; Nancy R. Cook, ScD; Christine M. Albert, MD, MPH; J. Michael Gaziano, MD, MPH; Julie E. Buring, ScD; JoAnn E. Manson, MD, DrPH
 

JAMA. 2008;300(17):2012-2021.

Folate, vitamin B₆, and vitamin B₁₂ are thought to play an important role in cancer prevention. To evaluate the effect of combined folic acid, vitamin B₆, and vitamin B₁₂ treatment on cancer risk in women at high risk for cardiovascular disease. In the Women's Antioxidant and Folic Acid Cardiovascular Study, 5442 US female health professionals aged 42 years or older, with preexisting cardiovascular disease or 3 or more coronary risk factors, were randomly assigned to receive either a daily combination of folic acid, vitamin B₆, and vitamin B₁₂ or a matching placebo. They were treated for 7.3 years from April 1998 through July 31, 2005.

Daily supplementation of a combination of 2.5 mg of folic acid, 50 mg of vitamin B₆, and 1 mg of vitamin B₁₂ (n = 2721) or placebo (n = 2721). A total of 379 women developed invasive cancer (187 in the active treatment group and 192 in the placebo group). Compared with placebo, women receiving the active treatment had similar risk of developing total invasive cancer (101.1/10 000 person-years for the active treatment group vs 104.3/10 000 person-years for placebo group; hazard ratio [HR], 0.97, breast cancer (37.8/10 000 person-years vs 45.6/10 000 person-years, respectively; HR, 0.83 or any cancer death (24.6/10 000 person-years vs 30.1/10 000 person-years, respectively; HR, 0.82.

Conclusion  Combined folic acid, vitamin B₆, and vitamin B₁₂ treatment had no significant effect on overall risk of total invasive cancer or breast cancer among women during the folic acid fortification era.

Folate, vitamin B₆, and vitamin B₁₂ (water-soluble, essential B vitamins) are important for DNA synthesis and methylation, critical processes for upholding DNA integrity and regulating gene expression, respectively, and are thus thought to play an important role in cancer prevention. Background fortification of the food supply with folic acid (a synthetic form of folate), a policy that began in the United States in 1998 to reduce risk of neural tube defects, has improved folate status in the general population.

Approximately one-third of US adults currently take multivitamin supplements containing folic acid, vitamin B₆, and vitamin B₁₂. Observational studies, which were conducted mostly before folic acid fortification, in general support an inverse association between high intake or blood level of folate, vitamin B₆, and vitamin B₁₂ and risk of cancer, particularly colorectal neoplasia and breast cancer, and primarily among those individuals consuming alcohol, a known antagonist for these B vitamins. Data from randomized trials of folic acid alone or in combination with B vitamins and cancer risk are limited, not entirely consistent, and 1 trial has even raised concerns about deleterious effects.

Women have been underrepresented in previous trials with B vitamins. We conducted a detailed analysis of cancer end points in the Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS), a large randomized trial designed to test the effect of combined folic acid, vitamin B₆, and vitamin B₁₂ on prevention of cardiovascular disease (CVD) among high-risk women during the folic acid fortification era.

In the WAFACS trial, up to 7.3 years of treatment with combined folic acid, vitamin B₆, and vitamin B₁₂ had no significant effect on overall risk of total invasive cancer, breast cancer, other individual cancers, or cancer death among women at high risk for CVD. There were no differences according to current use of multivitamin supplements, intakes of total folate, vitamin B₆, and vitamin B₁₂, or history of cancer at baseline. Lack of effect for total invasive cancer did not vary over time. We found a significant interaction by age group and a possible benefit among women aged 65 years or older at study entry.

Of the 5442 women, 3492 (64.2%) had a prior CVD event and 1950 (35.8%) had 3 or more coronary risk factors. In addition, 418 (7.7%) had a prior cancer event at baseline (diagnosed at least 10 years before enrollment), 307 (5.6%) consumed 15 g/d or more of alcohol, and 1247 (22.9%) were current users of multivitamin supplements. The mean (SD) values at baseline were 62.8 (8.8) years for age and 30.6 (6.7) for body mass index, calculated as weight in kilograms divided by height in meters squared. There were no significant differences in baseline characteristics between the active treatment group and the placebo group Three hundred seventy-nine women developed invasive cancer (187 in the active treatment group and 192 in the placebo group). Treatment with combined B vitamins had no significant effect on risk of total invasive cancer (101.1/10 000 person-years for the active treatment group vs 104.3/10 000 person-years for the placebo group; HR, 0.97 or individual cancer end points, including breast cancer (37.8/10 000 person-years vs 45.6/10 000 person-years, respectively; HR, 0.83 and colorectal cancer (9.7/10 000 person-years vs 12.0/10 000 person-years, respectively; HR, 0.81. There was no difference in any cancer death (24.6/10 000 person-years vs 30.1/10 000 person-years, respectively; HR, 0.82 or death from any cause. Cumulative incidence curves indicated no variation over time for total invasive cancer, whereas a decrease in breast cancer risk appeared to emerge after approximately 3 years. After excluding the first 2 years of follow-up as prespecified, the HRs were 0.94 (95% CI, 0.73-1.20) for total invasive cancer, 0.73 (95% CI, 0.50-1.06) for breast cancer, and 0.92 (95% CI, 0.53-1.60) for any cancer death.

In analyses censoring women at the time they stopped taking at least two-thirds of their study pills, the null results remained for total invasive cancer (148 vs 140 cases; HR, 1.04; 95% CI, 0.83-1.32), breast cancer (59 vs 64 cases; HR, 0.91; 95% CI, 0.64-1.30), and any cancer death (29 vs 36 cases; HR, 0.80; 95% CI, 0.49-1.30).

Age significantly modified the effect of combined B vitamin treatment on risk of total invasive cancer and breast cancer. A significantly reduced risk was observed for total invasive cancer (HR, 0.75) and breast cancer (HR, 0.62) among women aged 65 years or older at study entry, but no reductions in risk were observed among younger women (40-54 years or 55-64 years). There were no effect modifications by use of multivitamin supplements, intakes of total folate, vitamin B₆, and vitamin B₁₂, history of cancer, alcohol intake, or other risk factors at baseline. Finally, no significant effects were observed according to hormone receptor status or other characteristics of breast tumors, except that a borderline significant reduction was found for estrogen receptor–positive and progesterone receptor–negative tumors

There is a concern that increasing folate status resulting from mandatory folic acid fortification and supplementation may increase cancer risk in some people because folate may play a dual role in carcinogenesis—prevent tumor initiation when it is administered early in carcinogenesis and in individuals with suboptimal folate status, but promote tumor development when it is administered later in carcinogenesis (ie, once premalignant lesions are established) and in individuals with high folate intake. In the WAFACS trial, consistent with the implementation of US folic acid fortification policy, plasma folate levels were increased in the placebo group during 7.3 years of follow-up (8.9 vs 15.4 ng/mL). With the long duration of treatment and follow-up, the WAFACS trial showed no effect on cancer risk even among those women taking multivitamin supplements or with higher intake of total folate, vitamin B₆, and vitamin B₁₂, or with a history of cancer at baseline. Two other large randomized trials assessing folic acid, vitamin B₆, and vitamin B₁₂ in relation to CVD risk have reported cancer outcomes. In the Heart Outcomes Protection Evaluation (HOPE)-2 trial, which included 5522 patients aged 55 years or older who had vascular disease or diabetes, an average of 5 years of treatment with the same daily combination of 2.5 mg of folic acid, 50 mg of vitamin B₆, and 1 mg of vitamin B₁₂ had no significant effect on risk of total cancer (relative risk, 1.06; 95% CI, 0.91-1.23), cancers of breast, colon, lung, or prostate, or cancer death. In the HOPE-2 trial, 71.1% of participants were from countries with folic acid fortification. The NORVIT trial, which included 3749 men and women aged 30 to 85 years who recently had acute myocardial infarction in Norway, a country that has no mandatory folic acid fortification in foods, also reported no effect on cancer outcomes. Compared with the placebo group, there was no difference in cancer risk in those patients receiving the combined folic acid (0.8 mg), vitamin B₆ (40 mg), and vitamin B₁₂ (0.4 mg) treatment (40 vs 40 cases; relative risk, 1.02; 95% CI, 0.65-1.58) or combined folic acid (0.8 mg) and vitamin B₁₂ (0.4 mg) treatment (39 vs 40 cases) over an average of 3.3 years of treatment and follow-up.Also, in this trial, there was a suggestive beneficial effect on cancer risk in those patients treated with vitamin B₆ (40 mg) alone compared with those assigned to placebo (25 vs 40 cases). Taken together, data from randomized trials of combined B vitamins provide reassurance that folic acid supplementation up to a dose of 2.5 mg/d when combined with vitamin B₆ and vitamin B₁₂ does not appear to increase cancer occurrences and deaths among individuals at high risk for CVD during the folic acid fortification era. Because cancer has a long latency period, we cannot exclude the possibility that there might be beneficial or harmful effects of combined B vitamins on cancer that were not detectable within 7.3 years of treatment.

The results from randomized trials of folic acid alone and colorectal adenomas and of combined B vitamins and colorectal cancer have been mixed. In a trial that involved 60 men and women and was conducted before folic acid fortification, 24 months of treatment with 1 mg/d of folic acid nonsignificantly reduced the recurrence of colon adenomas. In another trial, which involved 1021 men and women and was conducted during folic acid fortification, 3 years of treatment with 1 mg/d of folic acid had no benefit on the recurrence of colorectal adenomas at 3 years of follow-up and at another 3 or 5 years of follow-up. Instead, there was an increased risk for advanced lesions at 3 years (relative risk, 1.32; 95% CI, 0.90-1.92) and at the second follow-up (relative risk, 1.67; 95% CI, 1.00-2.80). Those results did not differ significantly by sex, age, alcohol use, body mass index, or baseline plasma folate. In the third trial,which involved 945 men and women in England and was conducted before folic acid fortification, treatment with 0.5 mg/d of folic acid for 3 years had no effect on the recurrence of colorectal adenomas (relative risk, 1.07; 95% CI, 0.85-1.34) or advanced adenomas (relative risk, 0.98; 95% CI, 0.68-1.40). There was no increase in risk of colon cancer among women treated with combined B vitamins in the WAFACS trial, but a slightly increased risk was observed in the HOPE-2 trial.However, the number of colon cancer cases was limited in these randomized trials. The evidence for a beneficial effect of folate, vitamin B₆, and vitamin B₁₂ on colorectal cancer is strong in observational studies. Emerging data also suggest that vitamin B₆ may be more important than folate for colon cancer prevention in postmenopausal women. Thus, the effect of folic acid or combined B vitamins on colon cancer risk remains uncertain.

Lack of an overall effect for breast cancer in the WAFACS trial is generally consistent with observational studies that suggest no overall association between intake or blood level of folate, vitamin B₆, and vitamin B₁₂, and breast cancer risk. Data from several large cohort and case-control studies suggest that adequate folate may reduce breast cancer risk associated with alcohol intake or decrease risk of developing estrogen receptor–negative breast cancer. However, we observed no clear pattern of an effect according to alcohol intake and a borderline significant reduction was found for estrogen receptor–positive, progesterone receptor–negative breast cancer. The number of women consuming 15 g/d or more of alcohol or developing estrogen receptor–positive, progesterone receptor–negative or estrogen receptor–negative, progesterone receptor–negative breast cancers was limited in the WAFACS trial.

In 1 randomized trial of folic acid supplementation in pregnancy followed up for 36 years, women who received 0.2 or 5 mg of folic acid supplements during pregnancy had increased deaths from total cancer (n = 112) and breast cancer (n = 31); those women who received the higher dose of 5 mg had the highest risk. However, that study had short (several months in pregnancy) and remote (36 years ago) treatment and small number of breast cancer events. The possibility that the findings may be a result of chance and confounding cannot be excluded. The results from experimental studies have been mixed. Folate deficiency suppressed the N-methyl-N-nitrosourea–induced mammary tumors, while folate supplementation enhanced the initiation or early promotion of the N-methyl-N-nitrosourea–induced mammary tumors in rats in 1 study, but had no effect in 2 other studies.

In the WAFACS trial, a significant benefit of combined folic acid, vitamin B₆, and vitamin B₁₂ treatment was observed among women aged 65 years or older. If the finding is real and substantiated, the results may have public health significance because the incidence rates of cancer are high in elderly persons. The finding is biologically plausible because elderly individuals have increased requirements for these B vitamins. In addition, 2 large prospective investigations have shown that an inverse association between plasma vitamin B₆ and breast cancer risk is primarily present in postmenopausal women. In the HOPE-2 and NORVIT trials, there was no presentation by age for cancer end points, which were secondary outcomes. Because many subgroups were evaluated, we cannot exclude the possibility that the results from the subgroup analyses in the WAFACS trial are due to chance.

The strengths of the WAFACS trial include a randomized, double-blind, and placebo-controlled design, which minimizes confounding and bias that potentially affect the results from observational studies. To our knowledge, the WAFACS trial also had the longest duration of treatment with combined B vitamins of any trial to date, high follow-up rates, and relatively high adherence to treatment. Women have been underrepresented in other B vitamin trials, and the WAFACS trial is the largest trial of women. However, we cannot distinguish the effect attributable to any single B vitamin supplement compared with their combination. In addition, statistical power was insufficient to examine site-specific cancers. The WAFACS participants were female health professionals who tended to be health conscious, have well-balanced diets, and greater access to health care and screening, which may have led to lower occurrences of cancer. However, more than two-thirds of participants also were overweight or obese, and thus were at elevated risk for CVD and cancer. Therefore, the findings may not be directly generalizable to the entire US population. However, it seems unlikely that the exposure-disease relationships observed among women in the WAFACS differ from women in general.

In conclusion, treatment with combined folic acid, vitamin B₆, and vitamin B₁₂ provided neither beneficial nor harmful effects on overall risk of total cancer, breast cancer, or deaths from cancer among women at high risk for CVD