Sex hormone associations with breast cancer risk and the mediation of randomized trial postmenopausal hormone therapy effects

The Women’s Health Initiative (WHI) randomized, placebo-controlled postmenopausal hormone therapy (HT) trials were instrumental in leading to changes in clinical practice, including much reduced use of HT in the U.S. and around the world. Among the most important reasons for this change was the substantial elevation in breast cancer risk [1‐4] among women assigned to active hormones in the CEE + MPA trial. In contrast, a modest reduction in breast cancer incidence [5‐7] was observed among women assigned to active hormones in the CEE trial.

Considerable effort has been devoted to understanding these divergent breast cancer findings, through investigation of hazard ratio (HR) variations among trial cohort subsets [2, 6] and through comparisons of HRs with those from the companion WHI Observational Study. The latter analyses reveal higher HRs among women who started HT soon after menopause [8, 9], as has also been observed in other cohorts, for example, Beral et al. [10]. A recent report provides a detailed side-by-side presentation of CEE + MPA and CEE effects on breast cancer and other outcomes during the intervention and post-intervention phases of the WHI trials [11].

Serum sex hormone concentrations changed markedly following CEE or CEE + MPA administration in the WHI trial cohorts, with an approximate doubling of estradiol, estrone sulphate and sex hormone binding globulin (SHBG), and an approximate tripling of estrone with active treatment in each trial [12]. A recent report on a breast cancer nested case-control study within the CEE + MPA trial cohort noted that the treatment odds ratio was appreciably larger for women having relatively low baseline serum estrogens. For example, the OR (95% confidence interval (CI)) for breast cancer with CEE + MPA treatment was 2.47 (1.28, 4.79) among women in the lowest estradiol quartile, compared to 0.96 (0.44, 2.04) in the highest quartile [13].

Here we follow-up on this intriguing observation by examining the association between baseline serum sex hormones in the CEE + MPA and CEE trials and breast cancer risk, separately in the active treatment and placebo groups, during the intervention phases of the WHI trials. Concurrent associations of sex hormone changes from baseline to one year following randomization are also considered, and mediation analyses are conducted to examine the extent to which sex hormone changes can provide an explanation for the breast cancer findings in the two WHI trials.

Table 1 presents distributional information for breast cancer cases and controls, separately for the CEE and CEE + MPA trials, in relation to several baseline characteristics. The average age for cases was about 64 years, and average BMI was about 30.

Subsequent analyses exclude 31 breast cancer cases in the CEE + MPA trial and 16 cases in the CEE trial that occurred during the first year from randomization. The log-transformed ratios of year 1 to baseline values had approximately normal distributions for each sex hormone, but with a small fraction of outlying values, as anticipated from the blind duplicate data. Applying the interquartile range criterion (see Methods) led to the further exclusion of the following number of placebo cases or controls in the CEE + MPA trial: estradiol - 20, bioavailable estradiol - 25, estrone - 25, estrone sulfate - 28, SHBG - 21. Corresponding numbers in the active treatment group in the CEE + MPA trial were 11, 19, 11, 7 and 10; with the smaller number of exclusions consistent with the notion that measurement reliability is primarily an issue at low concentrations. The corresponding numbers of cases or controls excluded from the CEE trial placebo group on the basis of this IQR criterion were 31, 30, 17, 16 and 31, and from the CEE trial active treatment group were 9, 10, 5, 10 and 3.

Tables 2 and 3 show geometric means and 95% confidence intervals for cases and controls for each analyte, separately for the placebo and active groups in the CEE + MPA trial and the CEE trial. In the placebo group in either trial one sees the expected positive association between baseline or year 1 serum estrogens, and the inverse association of baseline SHBG, with disease risk. These same patterns prevail in the active treatment group in the CEE trial. In contrast, these patterns are not at all evident in the active treatment group in the CEE + MPA trial, suggesting that the use of CEE + MPA overrides the expected associations of baseline sex hormone concentrations with breast cancer risk.

Table 4 presents a refined version of the baseline sex hormone associations with breast cancer in the two HT trials. Odds ratios for a two-fold increment in baseline sex hormone level are presented from logistic regression analyses that control for various breast cancer risk factors (see Methods), in separate analyses for each analyte. Positive associations with most baseline estrogens and an inverse association with SHBG are evident in the placebo groups and in the active treatment group in the CEE trial. However, these associations are not evident in the active treatment group in the CEE + MPA trial.

Table 5 examines the ability of sex hormone changes from baseline to year 1 following randomization to mediate the divergent effects of the two hormone therapy regimens on breast cancer incidence. Odds ratios for a two-fold increment in baseline sex hormones are presented, along with odds ratios for a two-fold increment (‘change’) from baseline to year 1. The upper part of Table 5 includes bioavailable estradiol and SHBG jointly, while the lower part includes the other four serum estrogen measures simultaneously along with SHBG (bioavailable estradiol is calculated from the other measures and can not be included with them). The two parts of the Table provide a similar message: inclusion of the baseline to year 1 sex hormone changes in the regression model has little impact on the treatment odds ratio for CEE + MPA, but doing so appreciably increases the treatment OR for CEE toward the null. For example, in the lower part of Table 5, inclusion of the baseline to year 1 change variables in the analysis moves the estimated CEE + MPA treatment OR slightly away from the null, from 1.59 to 1.65, whereas the estimated CEE treatment OR moves substantially toward the null, from 0.71 to 0.92. While the upper part of Table 5 suggests that the SHBG increase with CEE may compensate somewhat for corresponding serum estrogen increases, the more detailed analysis in the lower part of Table 5 suggests that women having relatively large increases in serum estrone with CEE are at reduced breast cancer risk.

These analyses provide insight into the divergent breast cancer findings with CEE + MPA versus CEE in the WHI randomized, controlled trials. Specifically, women at a relatively low baseline breast cancer risk in the CEE trial continue at low risk if assigned to active treatment. Furthermore, the configuration of sex hormone changes among women assigned to active CEE provides an explanation for much of the observed breast cancer risk reduction (Table 5).

In sharp contrast, there is no evidence for any association between baseline sex hormone levels and breast cancer risk among women assigned to active treatment in the CEE + MPA trial, whereas associations are apparent in the placebo group (Table 4). Moreover, breast cancer risk does not relate to changes in sex hormones from baseline to year 1 in the CEE + MPA trial (Table 5). Evidently, in the presence of 2.5 mg/d medroxyprogesterone acetate, these risk variations are quite unimportant. Women having a favorable serum sex hormone profile, perhaps from years of good diet and activity patterns and normal weight maintenance, evidently have similar elevated breast cancer risk to those having an unfavorable serum sex hormone profile. These observations provide an explanation for the previously reported interaction between baseline sex hormone levels and CEE + MPA odds ratios [13].

The analyses presented here excluded about 10 to 15% of placebo group, and about 5% of active treatment group cases and controls based on an IQR outlier criterion as applied to the differences between baseline and one-year log-analyte concentrations. These exclusion rates are consistent with a poor correspondence between split sample quality control specimens for about 10% of samples, especially at low sex hormone concentrations. To ensure that our outlier exclusion method was not unduly influencing results, the Tables 2, 3, 4 and 5 analyses were repeated using Rosner’s [20] ‘many-outlier detection procedure’, which relies on departure from normality. This method resulted in fewer outlier exclusions, about 2 to 4% in the placebo groups and almost none in the active treatment groups. The analytic results with this less restrictive outlier detection approach were quite similar to those shown in Tables 2, 3, 4 and 5. A modest difference arose in the Table 5 analysis where the HR (95% CI) for a doubling of estrone from baseline to one-year was 0.68 (0.44, 1.05), as compared to 0.57 (0.35, 0.95) in Table 5.

The mechanisms whereby MPA would have such a dominant effect on breast cancer risk remain to be elucidated. MPA is a potent progestin with high affinity for progesterone and androgen receptors, and with little estrogen receptor affinity. It circulates in a form that is 88% bound to albumin, and is not bound by SHBG [21]. MPA could exert direct intracellular effects on division rates of breast ductal epithelial cells in a manner that achieves a high cell division rate regardless of the other sex hormone concentrations. A related possibility is that MPA sensitizes the proliferative response to low doses of estradiol [13].

The analyses presented here suggest that the collective changes in sex hormones with CEE contribute strongly to the observed breast cancer risk reduction [5‐7] in the WHI trial. It is plausible that the roughly two-fold increase in SHBG with CEE substantially offsets the corresponding large increase in serum estrogens in their effects on breast ductal epithelial cells. The upper portion of Table 5 provides some modest support for this line of reasoning, and may raise concerns about potential breast cancer risks with the trend toward use of transdermal estradiol, which does not materially increase SHBG in a first-pass hepatic effect, but substantially increases serum estradiol. Moreover, both oral and transdermal estradiols increase serum estradiol to a greater extent than does CEE.

The detailed analyses shown in the lower portion of Table 5 suggest that an additional mechanism may be at play with CEE. Specifically, women having a relatively large estrone increase with CEE use have a reduced (P = 0.03) breast cancer risk. This observation could align with the intriguing hypothesis that estrogen exposure following a sufficient period of estrogen deprivation induces apoptosis of nascent breast tumor cells [22]. Note, however, that the increases in SHBG and serum estrogens with CEE use are highly correlated (for example, correlations (95% CIs) of 0.73 (0.66, 0.79) between (log-transformed) estradiol and estrone changes; and 0.59 (0.59, 0.67) between estrone and SHBG changes in the control group women) making it challenging to distinguish among the changes in specific sex hormones in relation to breast cancer risk. The fact that the estrone change stands out in Table 5, in spite of these statistical challenges, is interesting and merits replication in other settings.

The strengths of this study are the randomized, controlled design of the WHI hormone therapy trials of adequate size, the quality sex hormone data assessment, and the ability to study CEE and CEE + MPA results side-by-side. Weaknesses include the absence of measurements on the biological changes resulting from the use of MPA.

In summary, post-treatment changes in serum estrogens and SHBG concentrations, or changes in the association of such concentrations with disease risk, have the potential to substantially explain both the elevation in breast cancer risk with CEE + MPA and the reduction in risk with CEE. The CEE data suggest that a roughly two-fold increase in SHBG may offset risk that may otherwise attend corresponding major serum estrogen increases, and also support the observation [9] that estrogen exposure following a sustained period of estrogen deprivation reduces risk [22]. However, when MPA is added to the hormone therapy regimen, the risk variations otherwise associated with these sex hormone levels are no longer evident, and an important increase in breast cancer risk follows, especially among women who would otherwise be at relatively low risk. Whether different formulations or routes of delivery of HT will provide similar results is unknown and also warrants further study.