Carotenoids, Retinol, and Vitamin E and Risk of Proliferative Benign Breast Disease and Breast Cancer

Carotenoids, Retinol, and Vitamin E and Risk of Proliferative Benign Breast Disease andBreast CancerAuthor(s): Stephanie J. London, Evan A. Stein, I. Craig Henderson, Meir J. Stampfer, WilliamC. Wood, Steven Remine, Jan R. Dmochowski, Nicholas J. Robert and Walter C. WillettSource: Cancer Causes & Control, Vol. 3, No. 6 (Nov., 1992), pp. 503-512Published by: SpringerStable URL: http://www.jstor.org/stable/3552858 .

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Cancer Causes and Control, 3, 503 - 512

Carotenoids, retinol, and vitamin E and risk of proliferative benign

breast disease and breast cancer

Stephanie J. London, Evan A. Stein, I. Craig Henderson, Meir J. Stampfer, William C. Wood, Steven Remine, Jan R. Dmochowski, Nicholas J. Robert, and Walter C. Willett

(Received 26 May 1992; accepted in revisedform 4 August 1992)

We investigated the relationship between serum levels of retinol, 3-carotene, a-carotene, lycopene, a-tocopherol, and y-tocopherol as well as intakes of retinol, carotene, and vitamin E and the risks of breast cancer and proliferative benign breast disease (BBD) in a case-control study of postmenopausal women in the Bos- ton, MA (United States) area. Serum nutrient data were available for 377 women with newly diagnosed stage I or II breast cancer and 173 women with proliferative BBD. Controls were 403 women who were evaluated at the same institutions but did not require a breast biopsy or whose biopsy revealed nonproliferative BBD. We observed no significant associations between serum levels of these micronutrients and risk of proliferative BBD or breast cancer. The risk of breast cancer was decreased among women in the highest quintile of intake of vitamin E from food sources only (odds ratio [OR] for the highest quintile = 0.4, 95 percent confidence interval [CI] = 0.2-0.9; P, trend across quintiles = 0.02) but less so for total vitamin E intake including supplements (OR = 0.7, CI = 0.4-1.3; P, trend = 0.07).

Key words: a-carotene, a-tocopherol, benign breast disease, 3-carotene, breast cancer, dietary intake, y-tocopherol, lycopene, serum, United States.

Introduction Vitamin A and related compounds play an important role in cell differentiation and, therefore, are hypothe- sized to influence carcinogenesis.' Dietary carotenoids, which include precursors of vitamin A, also may

mediate cancer risk by this mechanism or, as with vitamin E, by antioxidant properties.2 Extensive experimental evidence indicates that certain natural and synthetic retinoids inhibit mammary carcinogenesis in

Dr London is with the Department of Preventive Medicine, University of Southern California, Los Angeles, CA, USA. Dr Stein is with Medical Research Laboratories, Cincinnati, OH, USA. Dr Henderson was with the Dana Farber Cancer Center and the Department of Medicine, Harvard Medical School, Boston, MA, USA, and is currently with the University of California at San Francisco, San Francisco, CA, USA. Drs Stampfer and Willett are with the Department of Epidemiology, Harvard School of Public Health, Boston, MA, and the Channing Laboratory, Department of Medicine at Brigham and Women's Hospital, Harvard Medical School, Boston, MA. Dr Willett is also with the Department of Nutrition, Harvard School of Public Health, Boston, MA. Dr Wood was with the Department of Surgery, Massachusetts General Hospital, Boston, MA, and is currently with the Department of Surgery, Emory University, Atlanta, GA, USA. Dr Remine was with the Department of Surgery, Lahey Clinic, Burlington, MA, and is currently with Health Cleveland, Fairview General Hospital Cleveland, OH. Dr Robert was with the Department of Medicine, New England Medical Center, Tufts University, Boston, MA, and is currently with the Division ofHematol- ogy/Oncology, Fairfax Hospital, Fall Church, VA, USA. Dr Dmochowski is with the Department of Surgery, Lahey Clinic, Burlington, MA. Address correspondence to Dr Stephanie London, USC School of Medicine, PMB B306, 1420 San Pablo St, Los Angeles, CA 90033, USA. This project was supported by research grant CA 40429 from the National Cancer Institute. Dr London was supported in part by an Institutional National Research Service Award (CA 09001)from the National Cancer Institute.

? 1992 Rapid Communications of Oxford Ltd Cancer Causes and Control. Vol 3. 1992 503



S. J. London et al

rats.' However, there are few experimental data on whether carotenoids, precursors of vitamin A, have their own activity against mammary carcinogenesis. Experimental data on the role of vitamin E in mammary carcinogenesis in rodents are conflicting."-

Epidemiologic studies of retinol and carotenoid intake in relation to breast cancer risk are inconclusive. A recent pooled analysis of case-control studies revealed a modest inverse association between the intake of 3-carotene, but not retinol, and breast cancer risk among postmenopausal women.6 Results of more recent studies are inconsistent for both retinol and

3-carotene.7-'5 Weak inverse associations between breast cancer risk and intakes of vitamin A and carotene were found in the two cohort studies.""7 There are fewer epidemiologic studies of vitamin E intake in relation to breast cancer risk and results are inconclusive.7.10..1416

Because recall bias may influence case-control studies of micronutrient intake, the use of objective measures of nutritional status may be more informa- tive. Human data on blood levels of retinol, carotenoids, and tocopherols in relation to breast cancer risk are sparse and inconsistent.9,?'0•,s8-2' We have found no

published data on breast cancer risk in relation to blood levels of other carotenoids such as a-carotene or lycopene.

Inverse associations between intakes of preformed vitamin A and carotene and proliferative benign breast disease (BBD) have been found in two studies.2- The presence of proliferative BBD increases subsequent breast cancer risk.24

To address the associations between serum levels of retinol, carotenoids (3-carotene, a-carotene, and lycopene), and vitamin E, and the risk of breast cancer as well as proliferative BBD, we analyzed data from a case-control study of women being evaluated for breast abnormalities at five Boston-area (MA, United

States) hospitals. We also examined the relationship between intakes of retinol, carotene, and vitamin E-as assessed by a semiquantitative food-frequency questionnaire-and the risk of breast cancer and proliferative BBD.

Methods

Cases were postmenopausal women diagnosed with stage I or II breast cancer between 1986 and 1988 at five teaching hospitals in the Boston area. Postmenopausal women who sought evaluation for a breast abnor- mality, mammography, or breast symptoms at the same institutions during the same time period, but who were not diagnosed as having breast cancer, were enrolled as noncases. Subjects with a prior history of

cancer (exclusive of nonmelanoma skin cancer) were excluded.

Noncases were classified according to the outcome of their breast evaluation. A research nurse reviewed the pathology reports for all subjects with a breast biopsy. Based on the histologic features recorded in the pathology report, we classified women into three categories: nonproliferative disease; proliferative disease without atypia; and atypical hyperplasia.25 The category of proliferative disease without atypia included women with ductal hyperplasia, without atypia, as well as sclerosing adenosis and papillomatosis. The category of atypical hyperplasia included women with ductal or lobular atypical hyperplasia. Women whose biopsies revealed none of these features were classified as having nonproliferative disease. These women, together with those who did not undergo a biopsy, served as controls.

We identified 1,502 potential subjects: 631 cases and 871 women without breast cancer (noncases). An additional 144 subjects declined to participate before we could ascertain whether they had breast cancer. A total of 402 cases and 597 noncases agreed to participate in the study. Assuming that the percentage of cases among subjects with known status is the same among the 144 subjects with unknown case or noncase status, then the response rate is 58.2 percent for cases and 62.5 percent for noncases.

Subjects were asked to complete a questionnaire regarding medical history and breast-cancer risk factors and then to return a semiquantitative food-frequency questionnaire by mail. Blood was collected into evacuated tubes without anticoagulant and placed into a cooler with ice until centrifugation at room tem- perature at 1,000 X g for 10 min. Serum was stored at - 700C for up to nine months until samples were shipped on dry ice to the participating laboratories for analysis. The mean time between the drawing of blood and freezing of serum was 4 h (standard deviation [SD] = 4 h) for both cases and noncases. Care was taken to minimize exposure of the samples to light.

Serum was analyzed for 13-carotene, a-carotene, retinol, lycopene, a-tocopherol, and y-tocopherol by simultaneous high-performance liquid chromatography at Medical Research Laboratories (Cincinnati, OH) with retinyl acetate, a-tocopherol acetate, and retinyl palmitate as internal standards.26 Cholesterol, high-density lipoprotein (HDL), and triglyceride analyses were performed by Damon Laboratories (Westwood, MA). Intra-assay and inter-assay coefficients of variation (CV) were assessed in blind replicate samples sent to participating laboratories along with samples on study subjects so that the laboratory per- sonnel were not aware that replicate samples were

504 Cancer Causes and Control. Vol 3. 1992



Carotenoids, tocopherols, and breast cancer risk

included. The intra-assay coefficients of variation were: 12.2 percent for retinol; 16.6 percent for 0-carotene; 18.6 percent for a-carotene; 10.5 percent for lycopene; 7.0 percent for a-tocopherol; 8.6 percent for y-tocopherol; 4.0 percent for cholesterol; 2.6 percent for triglycerides; and 1.4 percent for HDL cholesterol. Inter-assay coefficients of variation were less than 12.0 percent for all analytes.

Dietary intake was assessed by a semiquantitative food-frequency questionnaire containing 116 food items with a specified portion size. Participants were asked how often, on average, during the past year they had consumed each food-with nine response categories ranging from 'never' to 'six or more times per day.' They also were asked to report foods consumed habit- ually but not included on the list of 116 items. Women were asked to describe the type of fat ordinarily used for baking and for frying food and to write in the brand and type of cooking oil and form of margarine (stick or tub). The questionnaire was returned usually within one month of phlebotomy.

A research nurse reviewed the food frequency questionnaire upon return and clarified duplicate responses or multiple missing responses by telephone. Women who reported intakes of less than 800 kilocalories per day or greater than 4,500 kilocalories per day were asked to repeat the questionnaire. If the total caloric intake from the repeat questionnaire was between 800 and 4,500 kilocalories per day, the second questionnaire was used in the analyses. If not, that subject was excluded from the analysis of nutrient data.

An earlier version of the food frequency questionnaire with only 61 food items has been validated exten- sively in relation to 28 days of food records.27,2 For our analyses, we separated intake of vitamin A into three categories: dietary carotene (carotenoid precursors of vitamin A from foods); dietary retinol (preformed vitamin A from foods); and retinol supplements (preformed vitamin A from vitamin pills). Available food composition tables do not permit a precise estimate of specific carotenoids (e.g., P-carotene, a-carotene, lycopene). The values for dietary carotene that we derived from published food tables"2 represent most of the p-carotene plus about half of the a-carotene and a small fraction of lycopene since these fractions approximate the vitamin A activity of the respective carotenoids. No subjects reported the use of p-carotene supplements. Estimates of carotene and a-tocopherol intake from the questionnaire were correlated reasonably well with plasma levels.30 The food and vitamin supplement items on the questionnaire account for over 90 percent of the between-person variance in intake of preformed vitamin A and p-carotene as deter- mined from 28 days of food records.?'

Among the 402 cases and 597 noncases who com- pleted a risk factor questionnaire, the blood sample was inadequate to measure carotenoids, retinol, and tocopherols for 25 cases and 21 noncases. These subjects were excluded, leaving 377 cases and 576 noncases (including 121 women with proliferative BBD without atypia, 52 women with atypical hyperplasia, and 403 control women who did not require a biopsy or whose biopsy revealed normal tissue or nonproliferative BBD) as the basic population for analysis of serum nutrients. The analysis of nutrient intakes excludes women who did not return a food frequency questionnaire (52 cases and 54 noncases) and women whose daily caloric intake estimated from the food frequency questionnaire was less than 800 kilocalories or greater than 4,500 kilocalories (12 cases and 13 noncases). This left 313 cases and 509 noncases (including 115 women with proliferative BBD without atypia, 45 women with atypical hyperplasia, and 349 control women who did not require a biopsy or whose biopsy revealed normal tissue or nonproliferative BBD) for the analyses of nutrient intake.

To minimize possible effects of breast cancer on levels of serum nutrients, we attempted to enroll subjects either before or as soon as possible after diagnostic evaluation for BBD or breast cancer. Among the 377 cases and 576 noncases with serum nutrient data, at the time of enrollment, 203 cases and 106 noncases were within six weeks of biopsy, 76 cases and 159 noncases were within six to 12 weeks from biopsy, 62 cases and 120 noncases were 12 to 24 weeks from biopsy, and three cases and 18 noncases were enrolled more than 24 weeks after biopsy. Twenty cases and one noncase were enrolled prior to breast biopsy. One hundred and forty-eight noncases did not undergo biopsy. For three cases, the date of biopsy was not recorded--these subjects were enrolled within 69 days of mastectomy.

The data were analyzed using unconditional logistic regression.2 Because women with proliferative BBD, with and without atypia, are at increased risk for breast cancer24 and intake of preformed vitamin A and carotenoids might decrease the risk of these lesions,22•' we calculated separate odds ratios (OR) for three outcomes: proliferative BBD without atypia; atypical hyperplasia; and breast cancer. Women with nonproliferative BBD and women whose evaluation did not result in a biopsy served as the control group for all analyses.

The distribution of serum and dietary nutrients among all subjects was used to assign quintiles. We adjusted serum values for carotenoids, retinol, and tocopherols for serum cholesterol, HDL, and triglycerides by regressing the serum micronutrients on the serum lipids (after natural logarithmic transformation)

Cancer Causes and Control. Vol 3. 1992 505



S. J. London et al

and then including the resulting residuals in the logistic regression models. For the 16 women missing lipid values, the group mean for that analyte was used in the regression analysis. Caloric intake, after logarithmic transformation, was included in models of nutrient intakes as a continuous variable to adjust for caloric intake. Tests for trend were done using ordinal variables.

Results The medians, 20th and 80th percentiles of age, serum concentrations, and dietary intakes of retinol, carotenoids, and tocopherol are shown in Table 1. No sub- stantial differences are evident across the four outcome categories. The relations between proliferative disease without atypia, atypical hyperplasia, and breast cancer, and traditional risk factors for breast cancer are presented in Table 2. Since controls were women who sought evaluation for a benign breast condition, they were more likely to have had a breast biopsy in the past than were the cases. Thus, prior history of BBD was not a risk factor in these data.

The association with family history may have been attenuated by a greater tendency of women with a family history to seek breast evaluation. We observed an increased risk with later age at first birth but no association with parity. Weight five years prior to

enrollment was associated somewhat more strongly with breast cancer risk than body mass index. Alcohol intake was not associated with breast cancer risk in this population of women who reported relatively light drinking (data not shown). We observed no clear associations between proliferative disease without atypia or atypical hyperplasia and any of these risk factors for breast cancer. In addition to adjusting for age, we adjusted all results for the variables in Table 2 and alcohol intake. However, none of the associations were altered materially by this adjustment.

The ORs for quintiles of serum concentrations of a-carotene, p-carotene, lycopene, retinol, and a-tocopherol in relation to each of the three outcomes after adjustment for breast-cancer risk factors are presented in Table 3. There were no clear associations between any of the analytes measured and risk of proliferative BBD, with or without atypia, or breast cancer.

The concentrations of y-tocopherol and a-tocopherol in the serum were correlated negatively (Pear- son r = - 0.29, P < 0.0001). When a- and y--tocopherol were entered simultaneously into a logistic regression model, women in the highest quintile of either tocopherol were at decreased risk of proliferative disease without atypia and breast cancer but neither trend was statistically significant (data not shown).

We examined the relationship between intakes of carotene, retinol (with and without supplements), and

Table 1. Medians, 20th and 80th percentiles of serum concentrations and dietary intakes of selected nutrients among postmenopausal women in the Greater Boston Area, 1986-88

Category of breast pathology

Nonproliferative Proliferative Atypical Breast or no biopsy without atypia hyperplasia cancer

Age 62 (56-68)a 61 (55-65) 63 (59-68) 64 (58-69)

Serum nutrient Number of subjects 403 121 52 377 a-carotene (nmol/1) 66.10 (26.1-147.2) 55.90 (27.9-113.6) 55.00 (26.1-119.2) 65.20 (27.9-147.2)

3-carotene (i?mol/l) 0.44 (0125-0.78) 0.42 (0.23-0.68) 0.45 (0.25-0.61) 0.44 (0.25-0.76) Lycopene (pmol/l) 0.60 (0.33-0.91) 0.57 (0.34-0.82) 0.57 (0.39-1.07) 0.57 (0.30-0.91) Retinol (?pmol/l) 1.99 (1.64-2.44) 1.92 (1.53-2.44) 2.04 (1.64-2.55) 2.02 (1.61-2.48) a-tocopherol (pmol/l) 30.00 (23 -42) 30.00 (22-40) 30.00 (25-36) 29.00 (23-40)

Daily dietary intake Number of subjects 349 115 45 313 Carotene (IU) 8,855 (4,125-15,685) 9,305 (6,003-15,751) 8,357 (3,963- 13,550) 8,788 (3,818-13,981) Retinol, total (IU) 2,765 (589-6,872) 2,575 (904-6,301) 2,558 (740-5,955) 2,277 (509-6,292) Retinol, food only (IU) 1,538 (498-3,482) 1,517 (566-3,677) 1,443 (699-2,558) 1,573 (451-3,162) Vitamin E, total (mg) 11.1 (5.0-36.7) 10.0 (7.0-35.9) 9.2 (5.1-23.0) 9.5 (4.2-29.8) Vitamin E,

food only (mg) 8.0 (4.4-11.2) 8.4 (5.9-9.9) 7.3 (4.4-9.2) 7.6 (3.8-10.1)

Total energy intake (kcal) 1,707 (1,151-2,175) 1,821 (1,408-2,245) 1,699 (1,111-2,234) 1,737 (1,016-2,245)

- Median and, in parentheses, 20th-80th percentile.





vitamin E (with and without supplements), and the risk of proliferative BBD and breast cancer (Table 4). Women in the highest quintile of carotene intake had

nonsignificantly lower risk of atypical hyperplasia and breast cancer. The risk of all three outcomes was decreased for women in the highest quintile of intake of vitamin E from food sources but the trend was statisti-

cally significant only for breast cancer. Additional

adjustment for quintiles of intake of polyunsaturated, monounsaturated, and total fat intake did not alter materially the associations between vitamin E from food sources and the three outcomes. Women in the highest quintile of intake of vitamin E including supplements were at nonstatistically significant decreased risk of breast cancer. Because the association with vitamin E intake, including the use of supplements, was

Table 2. Age-adjusted odds ratios (OR) and 95% confidence intervals (CI) for proliferative benign breast disease (BBD) without atypia, atypical hyperplasia, and for breast cancer in relation to risk factors for breast cancer among 953 postmenopausal women in the Greater Boston Area, 1986-88

Variable Proliferative BBD Atypical hyperplasia Breast cancer without atypia (n = 52) (n = 377)

(n = 121)

OR (CI) OR (CI) OR (CI)

Age at first birth (years) Nulliparous 1.0 1.0 1.0 <21 0.6 (0.3-1.3) 1.4 (0.5-3.8) 1.1 (0.7-1.9) 21-24 0.9 (0.5-1.6) 0.6 (0.2-1.5) 1.3 (0.9-2.0) 25-28 0.6 (0.3-1.1) 1.2 (0.5- 2.8) 1.2 (0.7-1.8) 29 + 1.0 (0.5-1.9) 1.6 (0.6-4.0) 1.8 (1.1-2.9) P, trend (0.62) (0.48) (0.03)

Parity Nulliparous 1.0 1.0 1.0 I Birth 0.7 (0.3-1.8) 1.5 (0.5-4.5) 1.5 (0.9-2.6) 2 Births 0.7 (0.4-1.3) 0.9 (0.4-2.1) 1.3 (0.9-2.0) 3+ Births 0.8 (0.5-1.3) 1.1 (0.5-2.4) 1.2 (0.8-1.8) P, trend (0.42) (0.96) (0.50)

Family history (mother or sister) No 1.0 1.0 1.0 Yes 0.9 (0.5-1.6) 1.5 (0.7-3.0) 1.2 (0.8-1.8)

Age at menopause (years) < 41 1.0 1.0 1.0 41-46 1.1 (0.6-2.2) 1.3 (0.5-3.4) 1.5 (0.9-2.4) 47-49 1.4 (0.7-2.8) 1.3 (0.5-3.8) 1.5 (0.9-2.4) 50-53 0.8 (0.4-1.6) 1.2 (0.5-3.0) 1.2 (0.8-1.9) 54+ 1.3 (0.6-2.9) 0.8 (0.2-3.0) 2.4 (1.4-4.1) P, trend (0.89) (0.86) (0.05)

Weight five years before enrollment (lb) < 124 1.0 1.0 1.0 125-134 0.6 (0.3-1.1) 0.9 (0.4-2.4) 1.0 (0.6-1.7) 135-144 1.0 (0.4-1.8) 0.9 (0.4-2.5) 1.0 (0.6-1.7) 145-164 0.7 (0.4-1.3) 0.9 (0.4-2.1) 1.3 (0.8-2.1) 165+ 1.3 (0.7-2.6) 0.6 (0.2-1.9) 1.5 (0.9-2.5) P, trend (0.36) (0.42) (0.06)

Age at menarche (years) < 12 1.0 1.0 1.0 13 0.9 (0.6-1.5) 1.4 (0.7-2.8) 0.9 (0.7-1.3) 14+ 0.7 (0.4-1.2) 0.9 (0.5-2.0) 0.9 (0.6-1.2) P, trend (0.21) (0.94) (0.37)

History of BBD No 1.0 1.0 1.0 Yes 1.1 (0.4-1.4) 1.4 (0.7-2.6) 0.8 (0.5-1.4)




S. J. London et al

Table 3. Adjusted odds ratios (OR)a and 95% confidence interval (CI) for proliferative BBD without atypia, atypical hyperplasia, and for breast cancer by quintile of serum concentration of selected carotenoids, retinol, and a-tocopherol among 953

postmenopausal women in the Greater Boston Area, 1986-88

Quintile Proliferative BBD Atypical hyperplasia Breast cancer median without atypia (n = 52) (n = 377)

(n = 121) OR (CI) OR (CI) OR (CI)

a-carotene (nmol/1) 17.4 1.0 1.0 1.0 37.8 1.3 (0.7-2.6) 1.2 (0.5-3.2) 1.1 (0.7-1.8) 62.7 1.3 (0.6-2.5) 1.3 (0.5-3.4) 1.2 (0.8-2.0)

100.6 0.9 (0.4-1.8) 0.6 (0.2-1.9) 1.1 (0.7-1.8) 219.5 1.1 (0.5-2.2) 0.7 (0.2-1.9) 1.2 (0.7-1.9) P, trend (0.53) (0.28) (0.71)

a-carotene (pmol/l) 0.17 1.0 1.0 1.0 0.30 1.8 (0.9-3.6) 1.2 (0.4-3.3) 1.7 (1.1-2.9) 0.42 1.6 (0.8-3.2) 1.1 (0.4-2.9) 1.3 (0.8-2.1) 0.60 0.9 (0.4-2.0) 1.0 (0.4-2.6) 1.1 (0.7-1.9) 0.98 1.1 (0.6-2.4) 0.8 (0.3-2.2) 1.2 (0.7-1.9) P, trend (0.68) (0.57) (0.97)

Lycopene (p.mol/l) 0.22 1.0 1.0 1.0 0.43 1.0 (0.5-2.0) 2.2 (0.7-6.9) 1.0 (0.6-1.6) 0.58 1.0 (0.5 -2.0) 1.8 (0.5-5.7) 0.8 (0.5-1.3) 0.77 0.8 (0.4-1.5) 1.2 (0.4-4.0) 0.8 (0.5-1.4) 1.10 0.8 (0.4-1.6) 2.7 (0.9-8.5) 1.0 (0.7-1.7) P, trend (0.31) (0.30) (0.82)

Retinol (plmol/1) 1.47 1.0 1.0 1.0 1.78 0.7 (0.4-1.4) 0.9 (0.3-2.3) 0.8 (0.5-1.4) 2.00 0.7 (0.4-1.5) 0.7 (0.3-1.9) 0.9 (0.5-1.5) 2.26 0.5 (0.2-1.0) 0.6 (0.2-1.7) 1.0 (0.6-1.7) 2.70 0.7 (0.4-1.4) 1.1 (0.4-2.8) 1.0 (0.6-1.7) P, trend (0.20) (0.85) (0.76)

a-tocopherol (.Lmol/l) 22 1.0 1.0 1.0 26 0.5 (0.3-1.1) 1.6 (0.6-4.7) 0.9 (0.5-1.4) 29 0.8 (0.4-1.6) 1.3 (0.4-3.7) 1.0 (0.6-1.7) 34 0.9 (0.4-1.8) 1.7 (0.6-4.9) 0.8 (0.5-1.3) 48 0.7 (0.4-1.3) 1.1 (0.3-3.2) 0.8 (0.5-1.2) P, trend (0.66) (0.92) (0.19)

a OR = odds ratio for increasing quintiles relative to the lowest quintile. Four hundred and three women with nonproliferative BBD on biopsy or who were not referred for biopsy were used as the control group for all comparisons. ORs were adjusted for age, alcohol intake, age at first birth, parity, family history of breast cancer, age at menopause, age at menarche, body weight, and prior history of BBD. ORs adjusted for serum lipids-cholesterol, triglycerides, and HDL cholesterol.

weaker despite the higher intake of vitamin E among women using supplements, we examined the major food sources of vitamin E intake in these data. The two

major contributors to the between-person variation in

vitamin E intake were mayonnaise and sweet potatoes. When we examined mayonnaise intake in relation to breast cancer risk, after adjusting for total caloric intake and breast cancer risk factors, the OR was reduced only in the highest quintile of mayonnaise intake (OR = 0.5, 95 percent confidence interval [CI]

= 0.3-1.0; P, trend = 0.26). Sweet potato intake was not associated with breast cancer risk. The inverse association with vitamin E from foods only was attenuated

slightly by inclusion of mayonnaise intake in the model (OR for highest quintile = 0.5, CI = 0.3-1.0; P, trend = 0.06).

Although we limited the study to women with

recently diagnosed stage I or II breast cancer who had not yet undergone chemotherapy or radiation to minimize changes in nutritional status from disease and





Table 4. Adjusted odds ratios (OR) and 95% confidence intervals (CI) for proliferative benign breast disease (BBD) without atypia, atypical hyperplasia, and for breast cancer by quintile of dietary intake of carotene, retinol and vitamin E among 822 postmenopausal women in the Greater Boston Area, 1986-88

Quintile Proliferative BBD Atypical hyperplasia Breast cancer median without atypia (n = 45) (n = 313)

(n = 115)a

ORb (CI) OR (CI) OR (CI)

Carotene (IU) 4,303 1.0 1.0 1.0 6,490 2.0 (0.9-4.3) 1.0 (0.3-2.8) 1.0 (0.6-1.7) 8,911 2.0 (0.9-4.5) 1.0 (0.4-3.0) 1.2 (0.7-2.0)

12,797 2.8 (0.9-4.5) 0.6 (0.2-2.0) 1.1 (0.7-1.9) 19,970 1.5 (0.7-3.5) 0.7 (0.2-2.1) 0.6 (0.3-1.1) P, trend (0.44) (0.34) (0.17)

Retinol, total (IU) 639 1.0 1.0 1.0

1,205 1.1 (0.5-2.4) 4.8 (1.3-17.8) 1.2 (0.7-2.0) 2,373 1.1 (0.5-2.3) 2.3 (0.6-8.7) 0.8 (0.5-1.4) 4,861 1.5 (0.7-3.3) 3.0 (0.8-11.1) 0.9 (0.5-1.5)

10,916 0.8 (0.4-1.7) 1.3 (0.3-5.0) 0.7 (0.4-1.3) P, trend (0.84) (0.61) (0.14)

Retinol, food only (IU) 555 1.0 1.0 1.0 911 0.5 (0.2-1.0) 4.8 (1.2-19.4) 1.0 (0.6-1.7)

1,462 0.5 (0.2-1.1) 6.1 (1.5-25.3) 1.4 (0.8-2.4) 2,352 0.5 (0.2-1.0) 3.2 (0.8-13.3) 0.9 (0.5-1.6) 4,358 0.8 (0.4- 1.6) 1.0 (0.2- 5.2) 0.9 (0.5-1.6) P, trend (0.54) (0.52) (0.57)

Vitamin E, total (mg) 5.8 1.0 1.0 1.0 7.4 1.9 (0.8-4.2) 1.2 (0.4-3.6) 1.0 (0.6-1.7) 9.2 2.4 (1.1-5.6) 0.9 (0.3-3.0) 0.7 (0.4-1.3)

20.5 1.4 (0.6-3.3) 0.9 (0.3-2.8) 0.5 (0.3-1.0) 368.1 1.5 (0.7-3.4) 0.4 (0.1-1.5) 0.7 (0.4-1.3) P, trend (0.86) (0.15) (0.07)

Vitamin E, food only (mg) 5.4 1.0 1.0 1.0 6.7 1.6 (0.7-3.4) 1.8 (0.6-5.7) 0.8 (0.5- 1.4) 7.6 1.5 (0.7-3.4) 1.3 (0.4-4.1) 0.9 (0.5-1.5) 8.6 1.6 (0.7-3.7) 0.9 (0.3-3.3) 0.7 (0.4-1.2)

11.0 0.6 (0.2-1.6) 0.4 (0.1-2.0) 0.4 (0.2-0.9) P, trend (0.42) (0.16) (0.02)

a Of 953 subjects on whom blood and risk factor questionnaires were obtained, food-frequency questionnaire data were available on 822, therefore totals in all categories are lower than in Table 3.

b ORs for increasing quintiles relative to the lowest quintile. Women (349) with nonproliferative BBD on biopsy or who were not referred for biopsy served as the control group. ORs were adjusted for caloric intake, age, alcohol intake, age at first birth, parity, family history of breast cancer, age at menopause, age at menarche, prior history of benign breast disease, and body weight.

treatment, we examined the possibility that changes in concentrations of serum nutrients due to disease pro- gression might have influenced the results. We divided cases according to the time from diagnosis to enrollment using the midpoint of the distribution-33 days. There were no important differences in the associations of breast cancer risk and serum concentrations of the carotenoids or a-tocopherol between the two

groups (data not shown).

Discussion We observed no clear associations between serum levels of retinol, 3-carotene or a-carotene, lycopene, a-tocopherol or y-tocopherol and the risk of proliferative BBD without atypia, atypical hyperplasia, or breast cancer. Women in the highest quintile of intake of vitamin E without supplements were at decreased risk of all three outcomes but the trend was statistically significant only for breast cancer. Vitamin E intake




S. J. London et al

including supplements was associated less strongly with breast cancer risk.

Results of previous studies on blood measurements of nutrients and risk of breast cancer (Table 5) have not provided clear evidence for the hypothesis, suggested by experimental data in rodents, that vitamin A, its precursors, and vitamin E inhibit mammary carcinogenesis. In general, there are few data and all are limited by small numbers or methodologic difficulties. The strongest inverse association with 1-carotene was found in the hospital based case-control study of Potischman et al.l The OR for the highest quartile of plasma 3-carotene concentration was 0.3. However, that study included women with advanced-stage breast cancer in whom plasma levels were lower than women

with less advanced disease." Our study included only women with newly diagnosed stage I or II breast cancer in whom the disease would not be expected to have altered nutrient levels. No association was observed in the two other case-control studies.9,1o

Results of cohort studies,1821,33,34 all of which have been small, are inconsistent (Table 5). Coates et al20 observed a relative risk of 0.3 for women in the highest tertile of retinol intake. Wald et a119 originally reported a decreased risk for women with higher prediagnostic levels of 3-carotene and ca-tocopherol but not retinol, but later observed that the results might have been an artifact of differential degradation in specimens between cases and controls due to freezing and rethaw- ing.3 A subsequent study"' of the same cohort revealed

Table 5. Studies of blood levels of carotenoids, retinol, and/or vitamin E in relation to breast cancer risk

Authors, date Study design Cases/ Nutrients Evidence of association Lipid controls RR' or Mean P < 0.05?b adjusted?

difference

Potischman et al, Case-control; 83/113 Retinol 0.9 n y 1990 Stage = all; P3-carotene 0.3 y y

Controls = benign breast a-carotene 1.6 n y disease Lycopene 0.6 n y

Marubini et al, Case-control; 214/215 Retinol 2.0 y y 1988 Stage = I, II; 13-carotene 1.2 n y

Controls = hospital

Gerber et al, Case-control; 319/344 Retinol Case > control c n y 1990 Stage = I, II; 3-carotene Case > control n y

Controls = hospital Vitamin E 4.2 y y

Wald et al, 1984 Prospective 39/78 Retinol 0.8 n n

1-carotene 0.5 n n Vitamin E 0.5 y n

Willett et al, 1984 Prospective 14/31 Retinol Case > control n y 1-carotene Case > control n y Vitamin E Case < control n y

Coates et al, 1988 Prospective 21/39 Retinol 0.3 n n

Russell et al, 1988 Prospective 30/288 Retinol Case < control n n Vitamin E Case > control n n

Knekt et al, 1990 Prospective 67/123 Retinol 0.8 n y 1-carotene 3.3 y y

Knekt et al, 1991 Prospective 67/67 Vitamin E 0.7 n y

R RR = relative risk for the highest relative to the lowest category, except for Wald et al, 1984, where R is for highest quintile relative to all

subjects combined. b Where results are given as relative risks, this column refers to the P value for the test for trend across categories. Where the results were available only as the differences in means, this column refers to the P-value for that difference. c Case > control signifies that the mean for cases was greater than the mean for controls.





no association between blood levels of retinol or

cr-tocopherol and breast cancer risk. Willett et aPr observed no association between retinol, carotenoid, and a-tocopherol levels and breast cancer risk. Three of the cohort studies's-20 are limited by a lack of adjustment for blood lipids.

In all of the cohort and case-control studies, including the current study, a single blood measurement was used to classify nutrient levels. The utility of a single blood measurement of carotenoids, retinol, and tocopherol is limited by both laboratory error and variation within subjects over time." Given that the magnitude of the associations under investigation may be modest, the laboratory variability for some of the analytes substantially decreases the power to detect an effect, particularly if the variability among subjects is low. With regard to variation within subjects over time, levels of carotenoids, retinoids, and tocopherols in other tissues may reflect longer term intake than do blood levels. Adipose tissue levels of tocopherols appear to represent long-term status and the same may be true for

carotenoids.? Carotenoids, tocopherols,373" and retinol" can be measured in the subcutaneous adipose tissue. Adipose tissue for micronutrient measurements can be obtained conveniently by needle aspiration38 and may be superior to a single blood level, particularly in case-control studies.

While not conclusive, published data on dietary intake suggest a small protective effect of increased consumption of carotenoids on the risk of breast cancer. While consistent associations were not observed in each individual study, a pooled analysis of eight case- control studies with data on P-carotene intake revealed a relative risk of 0.81 for an 8,000 IU/day increase in intake among postmenopausal women.6 Although we did not observe a clear association with carotene intake in our data, if the regression coefficient for the relation between carotene intake and breast cancer from that pooled analysis is used in our data, an OR for breast cancer of 0.6 is predicted for our highest quintile of intake, identical to what we actually observed. Retinol intake was associated positively but nonsignificantly with breast cancer risk in the same pooled analysis.6 In four recently published case-control studies not included in the pooled analysis, no significant association was found between breast cancer risk and 13-carotene or preformed vitamin A intake.o-'"14 Strong inverse associations between breast cancer risk and

p13-carotene were reported in case-control studies by Lee et aP and Zaridze.'5 In the only two cohort studies, modest inverse associations were observed with preformed vitamin A and carotenoids.","7

Among the case-control studies of vitamin E intake in relation to breast cancer risk, no association was

found in three"7,1," and a nonstatistically significant, inverse association in another.' In the only prospective data on vitamin E intake,"6 an inverse association with vitamin E intake of only borderline statistical signifi- cance was observed, although the sample size was large. The literature suggests that the expected magnitude of the postulated association with these nutrients is weak and/or that errors in measurement of intake substantially attenuate the observed associations in most studies.

As in all case-control studies, selection and recall bias must be considered in our data. We examined the possibility that the women with a benign breast evaluation enrolled as noncases might have been more health conscious and, therefore, possibly more concerned with their diets. Noncases were more likely than cases to have had more than three mammograms (16.5 percent of noncases cf 10.8 percent of cases). Theoretically, this difference might reflect selection bias that could have produced an inverse association between breast cancer risk and reported intake of carotene and vitamin E. Although this same mechanism should have produced an inverse association with nutrient levels, we found no association with carotenoid levels and only a weak, nonsignificant association with vitamin E levels. Furthermore, adjustment for frequency of mammograms did not alter substantially any of the results. Recall bias might have contributed to an association with intake of nutrients not corroborated by serum levels in our data.

Our results are compatible with the existing literature which suggests that there may be a weak association between intake of carotene and vitamin E with breast cancer risk. The lack of clear associations with serum values may represent a true absence of association, but this cannot be certain without sampling tissues, such as adipose tissue, that better reflect long-term intake of micronutrients than does a single blood measurement, or assessing these relationships in large prospective studies.

Acknowledgements-The authors wish to acknowl- edge the expert assistance of Akiko Tomita for pro- gramming; Monica Rosales, in preparation of the manuscript; dietician Laura Sampson, and registered nurses Roberta Hayes and Barbara Kalinowski, for recruitment of the study participants.

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Carotenoids, Retinol, and Vitamin E and Risk of Proliferative Benign Breast Disease and Breast Cancer