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Keywords:

  • breast cancer;
  • mammotropic hormones;
  • cohort;
  • European Prospective Investigation into Cancer and Nutrition (EPIC);
  • permissive;
  • estrogens;
  • hormones

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In a case–control study nested within the Greek EPIC cohort, serum levels of estrone, estradiol, androstenedione, dehydroepiandosterone sulfate, testosterone and IGF-1 were measured for 29 breast cancer patients and 58 control women, matched for age and menopausal status. There was little difference in breast cancer risk when values of 4–6—as compared to values of 1–3—hormones were elevated, a finding arguing against a positive interaction among these hormones. Breast cancer risk, however, was significantly and substantially lower among women with levels of all hormones below the corresponding age- and menopausal-status-predicted means, compared to women with levels of at least 1 hormone above the predicted mean (odds ratio = 0.11 with 95% confidence interval 0.01–0.90; p = 0.04). Our results suggest that the studied mammotropic hormones may act as permissive factors for breast cancer occurrence, and that the levels of some of them above the mean suffice for sustaining growth of a developing tumor. A corollary is that studies of mammotropic hormones in relation to breast cancer risk may also need to focus on the lower end of the distributions of these growth-enhancing hormones. © 2005 Wiley-Liss, Inc.

Prospective studies on endogenous hormones in relation to breast cancer occurrence have been undertaken mostly among postmenopausal women, because, among them, timing of sampling in relation to the menstrual cycle is not an issue.1, 2 However, prospective studies among premenopausal women have also been undertaken.3, 4 Moreover, case–control investigations of variable sample size and characteristics have also been performed during the last 3 decades.5, 6 The collective evidence for most of the investigated mammotropic hormones1—including estrone, estradiol, androstenedione, dehydroepiandrosterone sulfate (DHEAS) and testosterone—as well as prolactin,2 insulin-like growth factor 1 (IGF-1)7 and perhaps, on the basis of evidence regarding hormone replacement treatment, even progesterone,8 suggests that they are positively associated with breast cancer risk. Only for adiponectin, there is evidence that it may be inversely associated with breast cancer risk.9, 10 No breast cancer model has successfully accommodated the positive association of most mammotropic hormones with the risk of this disease, although empirical and theoretical attempts have been made.11, 12 Mutual confounding is an unlikely explanation, since the degree of confounding depends on the strength of the association of the confounder with both the exposure and the outcome under study, and the strength of the association of any of the mammotropic hormones with breast cancer is weak.13, 14 The mammotropic hormones may act synergistically in an additive or a superadditive way, but no evidence to that effect has been presented.12 It is also possible that mammotropic hormones act, in a nonspecific way, as permissive factors for breast cancer progression, so that inhibition of the clinical occurrence of the tumor would require low levels in all or most of them. We have evaluated this hypothesis using data from the Greek component of the European Prospective Investigation into Cancer and Nutrition (EPIC).

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Participants

A cohort of 28,572 volunteers, men and women, throughout Greece were enrolled between February 1994 and August 1999, in the context of the Greek component of the EPIC study. EPIC is conducted in 23 research centers across 10 European countries under the coordination of the International Agency for Research on Cancer (IARC), with the purpose of investigating the role of biological, dietary, lifestyle and environmental factors in the etiology of cancer and other chronic diseases. Blood samples were collected from all participants, according to a standardized protocol, fractionated and aliquoted in plastic straws containing 0.5 ml of plasma, serum, erythrocytes or buffy coat. Samples were stored (in liquid nitrogen (−196°C)) locally and centrally at IARC. Details on the design and methods of the EPIC study and the Greek cohort have been described previously.15 All procedures were in line with the Helsinki declaration for human rights; all volunteers signed informed consent forms, and the study protocol was approved by ethical committees at IARC, and, for the Greek EPIC cohort, by the University of Athens Medical School.

Among the Greek EPIC participants, 16,618 were women, and, by November 2002, 36 among them were diagnosed with histologically confirmed breast cancer. For each breast cancer patient, 2 control women were chosen at random among all cohort members who were alive and free of cancer at the time of diagnosis of the breast cancer patient. Matching criteria included age (± 6 months), the time of the day (± 1 hr) and, for premenopausal women, phase of menstrual cycle at blood donation.

Estrone, estradiol, androstenedione, DHEAS, testosterone and IGF-1 were measured at IARC with methods previously described and indicated here.4, 16 For the present study, we did not examine progesterone, because most breast cancer patients are postmenopausal, and at this stage of life, progesterone levels are minimal. We also did not examine adiponectin, which appears to be inversely associated with breast cancer risk.9, 10 Eventually, measurements for all the studied hormones were available for 29 breast cancer patients and their 58 matched controls.

Hormone assays

All assays were performed by study personnel at IARC, who were blinded as to the case–control status of the study subjects.4, 16 Serum concentrations of testosterone and DHEAS were measured by radioimmunoassay (Immunotech, Marseilles, France). Serum androstenedione concentrations were measured by a radioimmunoassay that used a double-antibody system for the separation of free and bound antigen (Diagnostic Systems Laboratories, Webster, TX). Estradiol measurements were conducted using an assay from DiaSorin (Saluggia, Italy), and estrone was measured by an assay from Diagnostic Systems Laboratories. IGF-1 was measured by enzyme-linked immunosorbent assays (ELISA, DSL), and the protocol for IGF-1 included an acid–ethanol precipitation step to remove IGF-binding proteins. Samples pertaining to matched study subjects, with each matched set containing samples from 1 case (patient) and 2 matched control subjects, were always analyzed in the same analytical batch. Based on results obtained for the quality control samples for all measurements done at IARC, the mean intrabatch coefficients of variation were estimated to be 6.1% for DHEAS, 8.4% for testosterone, 5.8% for androstenedione, 4.9% for estrone, 3.2% for estradiol and 6.2% for IGF-1.4, 16 Estrone and estradiol were expressed in pg/ml, androstenedione and testosterone in ng/ml and DHEAS and IGF-1 in μg/ml.

Statistical analysis

All analyses were conducted using the STATA statistical software, version 7. After calculation of descriptive data, we regressed each of the 6 hormones on age and menopausal status and we calculated regression-predicted means for each hormone for each breast cancer patient and control woman. We then counted breast cancer patients and controls whose values were higher than the corresponding regression-predicted mean with respect to none of the hormones, 1, 2, 3, 4, 5 and all 6 of them. Subsequently, we calculated the odds ratios for breast cancer if a woman had (i) none of the studied hormones above the predicted mean or (ii) 4 or more of the studied hormones above the predicted mean, using as baseline, women who had 1, 2 or 3 of the studied hormones above the predicted mean. Exact chi-square test and chi-square test for trend were used as needed.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The levels of the measured hormones among cases and controls are shown in Table I. Levels are generally higher among cases than among controls. Table II shows the correlation matrix among the mammotropic hormones measured in the controls. There is a pattern of positive interrelations among these mammotropic hormones, and values above 0.260 are statistically significant (p < 0.05).

Table I. Age, Menopausal Status and Hormone Levels Among Women with Breast Cancer and Matched Healthy Control Women. The Greek Epic Study
 Cases (n = 29)Controls (n = 58)p from t-test
Age in years: mean (range)57 (41–71)57 (42–71) 
Menopausal status (pre/post)14/447/22 
Hormones: mean (SD)
 Estrone (pg/ml)102.44 (82.42)79.21 (64.69)0.19
 Estradiol (pg/ml)60.97 (52.02)46.42 (41.98)0.20
 Androstenedione (ng/ml)1.33 (0.95)1.08 (0.57)0.20
 DHEAS (μg/ml)136.86 (88.85)115.87 (71.91)0.28
 Testosterone (ng/ml)0.63 (0.34)0.48 (0.21)0.04
 IGF-1 (μg/ml)213.23 (82.68)206.96 (74.79)0.73
Table II. Correlation Coefficients Among the Studied Hormones in Healthy Control Women
 EstroneEstradiolAndrostenedioneDHEASTestosteroneIGF-1
  • 1

    Dehydroepiandosterone sulphate.

Estrone1.000     
Estradiol0.8031.000    
Androstenedione0.2500.2291.000   
DHEAS10.4060.2080.4891.000  
Testosterone0.3540.1730.5600.7861.000 
IGF-10.3210.2220.2130.2150.1671.000

Table III presents the distribution of breast cancer patients and control women according to the number of hormones with levels above the corresponding age-and-menopausal-status-predicted means. There is little difference in breast cancer risk when values of 4–6—as compared to values of 1–3—hormones are elevated. The risk, however, is significantly and substantially lower among women with levels of all mammotropic hormones below the corresponding predicted means, compared to (i) women with levels of at least 1 hormone above the predicted mean (odds ratio = 0.11 with 95% confidence interval 0.01–0.90; p = 0.04) or (ii) women with levels of 1–3 hormones above the predicted mean (odds ratio = 0.12 with 95% confidence interval 0.01–0.98; p = 0.05). Alternatively, there is a significantly (p = 0.04) increasing trend of the disease odds ratio across all 7 columns of Table III, but when the first column (which refers to women with levels of all hormones below the corresponding mean) is excluded, there is essentially no trend across the remaining 6 columns (p = 0.53).

Table III. Distribution of Breast Cancer Patients and Control Women According to the Number of Hormones1 with Levels Above the Corresponding Age-and- Menopausal-Status-Predicted Means
Number of hormonesZeroOneTwoThreeFourFiveSixTotal
  • 1

    The hormones examined were estrone, estradiol, androstenedione, dehydroepiandosterone sulphate, testosterone and IGF-1.

Cases157526329
Controls141071186258
Odds ratio (95% confidence interval)0.12 (0.01–0.98)Baseline1.13 (0.43–3.00)
p from χ2 for trendAcross all columns: 0.04Across all columns but the first: 0.53

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In a case–control study nested within a population cohort, we have found evidence that when the levels of all 6 of the measured mammotropic hormones are below the corresponding age-and-menopausal-status predicted means, the risk for breast cancer is significantly and considerably reduced, by more than 80% (Table III). In contrast, there is no evidence that when the levels of most of the measured hormones are above the corresponding predicted means, the risk for breast cancer is significantly or substantially elevated (Table III). Indeed, there is little evidence in Table III for a linear trend across all columns, but the “zero” column (p = 0.53), whereas the significant linear trend across all columns (p = 0.04) is generated by the very low ratio of cases to controls in the “zero” column.

The positive association of breast cancer risk with all the studied hormones (Table I) and the positive inter-relations among these hormones (Table II) would be expected to amplify contrasts in the tails of the distributions. In our data, there is no strong evidence with respect to the upper tail of the distribution (columns “4” to “6”). Low levels of all of the studied hormones, however, are associated with substantially reduced risk of this disease. In other words, breast cancer risk appears to be very low unless the levels of at least 1 of these hormones are above the average. The results of our study suggest that the measured mammotropic hormones may act as permissive factors for breast cancer occurrence, and that even moderate levels of any one of these hormones (and perhaps additional ones) could suffice for sustaining growth of a developing tumor. A corollary is that studies of mammotropic hormones in relation to breast cancer risk may also need to focus on the lower end of the distributions of these growth-enhancing hormones. Our findings are reminiscent of the paradigm concerning the relation of testosterone with prostate cancer.17, 18, 19 Although eunuchs are known not to develop prostate cancer and antiandrogen therapy is an established treatment option for prostate cancer, an association between circulating androgen levels and prostate cancer risk has not been documented.

An important limitation of our study is its small size. We realize that the sample size does not allow firm documentation that mammotropic hormones have a permissive role in the development of breast cancer, but the findings point to this direction. Another limitation of the study is that we have investigated only the hormones that had already been measured in the EPIC collaborative center, among both pre- and postmenopausal women, thus excluding progesterone, the levels of which are minimal among postmenopausal women. Notwithstanding these limitations, our results accommodate the puzzling phenomenon of positive associations of virtually all mammotropic hormones with breast cancer risk, and are compatible with many experimental systems involving permissive factors for tumor development.20, 21

If confirmed in larger studies, the implications of our findings could be considerable. They would provide a simple explanation of why so many exogenous and endogenous mammotropic hormones turn out to be positively associated with breast cancer risk. They would accommodate the limited success of hormonal ablation procedures in the treatment of breast cancer. And, they would contribute to our understanding of the natural history of breast cancer during the medium and late stages of disease progression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The European Prospective Investigation into Cancer and Nutrition (EPIC) is coordinated by the International Agency for Research on Cancer (World Health Organization) and supported by the Europe Against Cancer Program of the European Commission. The hormone determinations were performed at IARC. The Greek segment of the EPIC study is also supported by the Greek Ministry of Health and the Greek Ministry of Education, as well as, by a fellowship honoring Vasilios and Nafsika Tricha.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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