1 Epidemiology Section, Cancer Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI.
2 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA.
3 Obstetrics and Gynecology Epidemiology Center, Brigham and Womens Hospital, Harvard Medical School, Boston, MA.
4 Department of Obstetrics and Gynecology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI.
5 Division of Medical Genetics, Childrens Hospital of Los Angeles, University of Southern California, Los Angeles, CA.
Received for publication January 4, 2002; accepted for publication May 28, 2002.
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ABSTRACT |
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case-control studies; diet; galactose; lactose; lactose intolerance; milk; ovarian neoplasms; uridine diphosphate galactose
Abbreviations: Abbreviations: CI, confidence interval; GALT, galactose-1-phosphate uridyltransferase, Hb, hemoglobin.
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INTRODUCTION |
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An association of lactose intake and metabolism with the risk of ovarian cancer is biologically plausible. High concentrations of circulating galactose, which is separated from glucose during digestion of lactose, may impair ovarian feedback to the pituitary, increasing gonadotropin secretion. Excess gonadotropin stimulation of the ovaries has been reported in galactosemic women who are deficient in the galactose-1-phosphate uridyltransferase (GALT) enzyme. Hypogonadism or ovarian failure occurs frequently among women with classic galactosemia, an inherited polymorphism in the GALT gene (3). Cramer et al. (4) hypothesized that women with galactosemia may be at increased risk of ovarian cancer because an excess concentration of gonado-tropin, including luteinizing hormone and follicle-stimulating hormone, may increase estrogenic stimulation of ovarian inclusion cysts, resulting in proliferation of ovarian epithelium. In support of this hypothesis, Cramer et al. (4) reported significantly lower GALT activity in ovarian cancer cases than in community controls. A significant positive trend in risk associated with a ratio of lactose intake to GALT activity was also found.
Conversion of galactose to glucose via the Leloir pathway involves several enzymes, including galactokinase, GALT, and uridine diphosphate galactose 4'-epimerase (5, 6). Although metabolic disorders may arise from deficiencies in the activity of any of these enzymes, classic galactosemia is known to be caused by several polymorphisms in the GALT gene (5, 6). Among the missense polymorphisms that may alter GALT stability is the N314D variant of the Duarte allele, an A6G transition at nucleotide 940 of the GALT gene that results in a substitution of aspartate for asparagine at the amino acid 314 position of the GALT protein. Uncertainty still exists regarding the influence of diet and of other metabolic genes on the enzyme expression of N314D variants (5, 7). A recent case-control study conducted by Cramer et al. (7) failed to confirm the positive association of GALT levels with ovarian cancer risk, but homozygosity for the Asp314 variant allele was positively related to the odds ratio for disease, especially for endometrioid and clear cell ovarian cancer.
The objective of this case-control study was to examine the hypothesis that reduced GALT activity, an increased ratio of lactose intake to GALT activity, and the inheritance of the N314D polymorphism are positively associated with the risk of epithelial ovarian cancer.
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MATERIALS AND METHODS |
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Population controls were frequency matched to cases on the basis of ethnicity (e.g., Japanese), 5-year age group, and study site. Eligible controls had no history of ovarian cancer and at least one intact ovary. In Hawaii, the pool of controls was identified from lists of female Oahu residents who were interviewed by the Health Surveillance Program of the Hawaii Department of Health. Annually, the Department of Health identifies a 2 percent representative sample of all households in the state by using a sampling procedure modeled after that of the National Health Survey (10). The refusal rate for this survey is extremely low (<5 percent) because it is conducted under statutory provision. This source was supplemented by selecting women aged 65 years or older who were Health Care Financing Administration participants on Oahu; this second source represented approximately 2 percent of selected controls. In Los Angeles, more than 95 percent of the controls were selected on the basis of a neighborhood walk procedure, in which a control was sought by use of a systematic algorithm based on the address of the case (11). Lists obtained through the Health Care Financing Administration were used to supplement selection of older controls (aged 65 years or older), representing about 1 percent of selected controls. Interview information was obtained from 607 controls (67 percent of eligible women), 430 (71 percent) of whom supplied a blood specimen for analysis.
In initiating this study, we recognized the high costs of the proposed biologic assays and decided, a priori, to examine a subset of approximately 50 percent of study participants in each geographic area, stratified on ethnicity (Caucasian vs. Asian). We did not stratify on lactose intake or lactase persistence because of the reported lack of correlation with GALT activity (4). This analysis included a subset of 239 cases (47.7 percent Caucasian, 35.1 percent Asian, and 17.1 percent "other") and 244 controls (45.5 percent Caucasian, 40.2 percent Asian, and 14.3 percent "other") who were interviewed during the first 3.5 years of the investigation.
Interviews were conducted in subjects homes by trained interviewers according to a standard protocol, and each interview required about 2.5 hours to complete. On average, cases were interviewed within 4 months of diagnosis. The questionnaire covered reproductive and gynecologic history; use of oral contraceptives and other hormones; contraceptive history, including tubal ligation; medical history; anthropometric measurements; and other lifestyle practices such as smoking and diet. A food frequency questionnaire, modeled after instruments used in other studies (12), included a minimal set of foods that contribute at least 85 percent of the dietary components of interest for each ethnic group. Intake of alcoholic beverages and use of dietary supplements were also assessed. The nutrient content of foods was determined from a customized food composition database developed and maintained at the Cancer Research Center of Hawaii (12). The food composition data were compiled largely from US Department of Agriculture Handbook 8 (13, 14), with supplementation by laboratory analyses of foods, and other commercial publications (1517). In addition to values for energy and macronutrients, the database includes values for more than 90 other nutrients, including lactose, calcium, and vitamin D. Intake from supplements was quantified by using a database compiled from supplement labels and manufacturers information. Total nutrient intake was calculated as the sum of the nutrients obtained from foods and supplements.
All blood samples were processed within 12 hours of collection. The plasma was separated from the cells by centrifugation (4°C for 15 minutes, 1,800 x g). The red cells were washed three times with equal volumes of 0.9 percent sodium chloride before freezing (70°C). GALT activity was determined in hemolysates by a carbon 14 labeling method, which uses chromatography on O-diethylaminoethyl-cellulose paper, as described by Lee and Ng (18). DNA was purified from peripheral blood leukocytes by sodium dodecyl sulfate/proteinase K treatment and phenol/chloroform extraction. In this study, we examined a single GALT polymorphism: an A6G transition at nucleotide 940, which replaces Asp314 with aspartate. Genotyping for the N314D polymorphism was performed by polymerase chain reaction and enzyme restriction assays, as described previously (6). The average coefficient of variation for the GALT analysis was 16.0 percent (range, 1.833.8 percent) among duplicate specimens.
The odds ratios associated with different levels of the exposure variables were evaluated by unconditional multiple logistic regression modeling of case-control status (19). The exposures of interest included several dietary variables, GALT activity, the ratio of lactose intake to GALT activity (defined as log(lactose) x 10/GALT), and the N314D genetic polymorphism. All exposures were entered into the models initially as binary indicator variables representing levels of exposures, generally quartiles for continuous measures, on the basis of the distribution for cases and controls. Odds ratios and 95 percent confidence intervals were computed by exponentiating the coefficients (and the 95 percent confidence intervals) representing exposure levels. Adjustment variables included age (continuous), ethnicity (by indicator: Caucasian, Asian, other), study site (Hawaii vs. Los Angeles), education (continuous), oral contraceptive pill use (ever vs. never), parity (ever vs. never), tubal ligation (yes vs. no), and energy intake (continuous). The associations of the exposure variables with the risk of ovarian cancer were also modeled within various subgroups (e.g., ethnic group) to examine consistency. We performed a test for linear trend in the logit of risk by using the likelihood ratio test, comparing models with and without a trend variable. The trend variable was assigned the median of the quartile for continuous measures and 1, 2, and 3 for the gene variable representing 0, 1, and 2 variant alleles, respectively.
Logistic regression was used to explore the joint association between GALT activity and other variables by modeling each level of interaction between the pairs of variables; the reference category was subjects whose GALT activity was below the median (24.1 µmol per hour/g hemoglobin (Hb)) and who were "unexposed" (e.g., had never used oral contraceptive pills). We compared this model obtained by using the likelihood ratio test with one in which only the main effects were modeled. Exact confidence intervals for odds ratios were computed, conditioned on the number of events (20). Analysis of covariance (21) was used to compare GALT levels across subgroups, controlling for the adjustment variables specified in the previous paragraph. Covariate-adjusted means were computed at the mean covariate level.
The distributions of study subjects by demographic characteristics and important ovarian cancer risk factors were similar among the 558 cases and 607 controls who provided interview information and the 239 cases and 244 controls included in the GALT subset. In the interview-only analysis, we found an inverse association of years of education, number of full-term pregnancies, number of years of oral contraceptive pill use, and history of tubal ligation with risk and a positive association with risk for a family history of breast or ovarian cancer (8). The odds ratios were consistent with the results shown in table 1 for the subset of women included in the present analysis.
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RESULTS |
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DISCUSSION |
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Two other case-control studies of galactose metabolism in relation to ovarian cancer have shown null associations. Herrinton et al. (25) reported no association between lactose intake, lactase deficiency, or GALT activity and risk of ovarian cancer among 108 cases and 108 controls in western Washington State. Webb et al. (22) observed no association of ovarian cancer with lactose or galactose intake, GALT activity, or ratio of lactose intake to GALT activity in a study of 107 cases and 277 controls in Queensland, Australia.
Use of oral contraceptive pills reduces gonadotropin levels and inhibits the hypergonadotropic effects of galactose, according to Harlow et al. (26). Under this hypothesis, women at greatest risk of hypergonadotropic hypogonadism would benefit the most from oral contraceptive pill use. Indeed, our finding of a much stronger inverse association of oral contraceptive pill use among women with low GALT activity than among those with high GALT activity supports this notion.
Although Cramer et al. (7) found no relation between GALT activity and the odds ratios for ovarian cancer, they did report a significantly higher frequency of the N314D polymorphism in cases (19.4 percent heterozygous and 1.6 percent homozygous for the Asp314 allele) than in controls (17.1 percent heterozygous and 0.4 percent homozygous for the Asp314 allele). In a study of 206 ovarian cancer cases and 248 controls in southern England, Morland et al. (27) also found that more cases than controls were heterozygous (25.7 percent of cases, 16.9 percent of controls) or homozygous (1.5 percent of cases, 0.4 percent of controls) for the Asp314 variant allele. However, this association resulted entirely from differences in the frequency of the Asp314 allele among women with serous and undifferentiated ovarian cancers, not endometrioid and clear cell histologic types, as reported by Cramer et al. (7).
The Duarte (N314D) variant of the GALT gene is thought to be prevalent in 620 percent of the nongalactosemic population (28), although the frequency of the N314D allele may be as low as 2 percent in Japan (5). The higher prevalence of the N314D polymorphism and lower levels of GALT among Caucasian women than among Asian and Pacific Islander women in our study is consistent with the observed ethnic variation in ovarian cancer incidence in Hawaii. However, we found no differences in the frequency of heterozygosity (10.5 percent of cases, 13.5 percent of controls) or homozygosity (0.9 percent of cases, 0.0 percent of controls) for the N314D polymorphism between all cases and controls combined or within ethnic group, suggesting alternative explanations for the ethnic variation in ovarian cancer risk.
Cramer et al. (7) hypothesized that, on the basis of a relation between the N314D polymorphism and reduced GALT activity, the presence of this polymorphism would lead to an increased risk of ovarian cancer (29). These investigators reported that both cases and controls who had the N314D polymorphism had lower biochemical GALT activity (6). We also observed significantly lower GALT levels in women with the N314D polymorphism. However, it is now known that the GALT response to the N314D polymorphism is modified by other genes (28). Laboratory data have demonstrated an association of the N314D polymorphism with normal or increased erythrocyte GALT activity in Duarte 1 carriers but as much as 50 percent reduced GALT activity in Duarte 2 carriers (30, 31). The Duarte 1 and Duarte 2 variants of galactosemia differ regarding other base changes that are in linkage disequilibrium with the N314D polymorphism, such as the L218L polymorphism in exon 7, which coexists with the N314D polymorphism in Duarte 1 variants but not Duarte 2 variants. A recent polymorphism analysis of the GALT gene found a deletion of four nucleotides (-119/-116delGTCA) in the 5' promoter region in Duarte 2 alleles but not in Duarte 1 alleles (31). This polymorphism may be functionally related to reduced GALT activity in Duarte 2 carriers. As in other disease processes, such gene-gene interactions on risk are complex and can be addressed only in carefully designed molecular studies with analyses of multiple, related genetic polymorphisms in large samples of women.
Strengths of the present study include a comprehensive, in-person interview; a population-based design; and a multiethnic composition of study subjects. This research was entirely hypothesis driven, but we did not commit our entire pool of subjects to the biologic analyses because of the high costs of the assays. Nonetheless, information regarding GALT activity and N314D genotype was available for at least 200 cases and controls, enabling us to test our hypotheses with reasonable study power.
Results of the analysis of the dietary information obtained in this study, including the inverse association of lactose intake with the risk of ovarian cancer, have been described previously (8). Regarding lactose, reasons for findings that were inconsistent with those from previous reports include 1) an inappropriate time frame for the dietary assessment and 2) bias in the exposure assessment or in selection of controls. We focused on recent diet in this study because of the difficulty in evaluating eating patterns in the distant past. It is conceivable that this was not the relevant time period for ovarian carcinogenesis, although our diet reference period was consistent with that of other studies (1, 4, 7, 22, 2426). It is unlikely that cases or controls would have systematically biased their responses regarding dairy intake, and our dietary assessment methodology was valid and reproducible (32, 33).
The level of participation in blood sample collection by interviewed cases (79 percent) and controls (71 percent) was good, although less so when considered as a percentage of eligible women: 48 percent of eligible cases and 47 percent of eligible controls provided a blood specimen. Low participation rates might have biased our findings if the biologic markers under study were a determinant of participation. We have no reason to think that this was the case. The odds ratios associated with pregnancy, oral contraceptive pill use, and other confounders were similar for subjects participating in the interview only and those participating in blood sample collection.
Misclassification of exposure or disease is an important source of concern, and we spent considerable time validating our assessment methods. Many of the exposures evaluated in our questionnaire, such as pregnancy and oral contraceptive pill use, were probably measured without much error. The possibility of such bias was minimized by using a structured questionnaire with well-defined probing methods and thorough training of the interviewers. To avoid disease misclassification, differentiation of primary ovarian cancer from metastatic colorectal cancer and from primary peritoneal carcinoma was achieved by close interaction of the pathologists with the clinicians and by correlation of the clinical record with pathologic review. Women who did not have definite, primary epithelial ovarian cancer according to clinical or microscopic evaluation were excluded from the study. Loss of cases because of illness or death (6 percent of eligible subjects) was minimized through the rapid-reporting system of the cancer registries in Hawaii and Los Angeles.
In conclusion, we found little evidence to support the hypothesis that either GALT activity or N314D genotype is a risk factor for ovarian cancer. The inverse association of oral contraceptive pill use with the odds ratios for ovarian cancer was strongest among women with low GALT activity, suggesting that gonadotropin levels may modify the beneficial effects of oral contraceptive pills on ovarian carcinogenesis. Long-term cohort studies that can evaluate the joint association of endogenous hormone levels and oral contraceptive pill use with the risk of ovarian cancer are needed to address this interaction more completely.
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ACKNOWLEDGMENTS |
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The authors thank the physicians, administrators, and cancer registrars at the following institutions in Honolulu, Hawaii, for their support of this study: Castle Memorial Hospital, Kaiser Foundation Hospital, Kapiolani Medical Location for Women and Children, Kuakini Medical Center, Queens Medical Center, Straub Clinic and Hospital, St. Francis Hospital, Tripler Army Hospital, and Wahiawa General Hospital.
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NOTES |
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REFERENCES |
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