In this large prospective study of 2-OH and 16α-OH estrone metabolites and breast cancer risk, we did not observe any significant associations overall with either individual metabolite or with the ratio of the two metabolites. Although we observed significant positive associations of both 2-OH estrone and the 2:16α-OH estrone ratio with ER-/PR-tumors, these results should be interpreted with caution given the small number of ER-/PR- tumors and that we are the first, to our knowledge, to report such an association. In addition, though we observed a significant positive association between the 2:16α-OH estrone ratio and breast cancer risk among lean women, the differences observed by BMI were not statistically significant.
The reproducibility of these estrogen metabolites is comparable to or better than other biomarkers with well established relationships to disease outcomes in epidemiologic studies, such as cholesterol (ICC=0.65) (50
) and blood pressure (ICC=0.60-0.64) (51
), as well as estradiol (0.68), estrone (0.74), and estrone sulfate (0.75) (47
) which have been consistently associated with breast cancer risk in this and other populations (1
). Thus, the lack of observed associations likely is not a result of poor reproducibility of a single measure of these metabolites.
To date, several epidemiologic studies have examined the association between 2-OH estrone and 16α-OH estrone and breast cancer risk. Several retrospective case-control studies have produced conflicting results, though the analysis of hormone levels after diagnosis, which may reflect tumor-driven activity, is a limitation of these studies (52
). Five prospective studies of eithaer urinary (29
) or serum (32
) estrogen metabolites among postmenopausal women have been published to date, with case numbers ranging from 42 (29
) to 272 (32
) among women who were not using postmenopausal hormones. No significant associations have been observed between 2-OH estrone and breast cancer risk, with RRs ranging from 0.80 (33
) to 1.61 (30
) for the top vs. bottom quartile or quintile or a doubling of 2-OH estrone concentration (comparable to our top vs. bottom quartile comparison). Three (30
) of four (30
) studies observed RRs above 1 for the association between 16α-OH estrone and breast cancer risk (range of RRs=1.23-2.47); none of the point estimates was statistically significant though one trend was suggestive (top vs. bottom quartile RR=2.47, 95% CI (0.90-6.80), p=0.06) (33
). No significant associations have been observed with the 2:16α-OH estrone ratio, with two studies reporting point estimates below 1 and two reporting estimates above 1 (range of RRs=0.71-1.31) (29
). Thus, similar to our overall findings, previous prospective studies have not observed any significant associations with either 2-OH or 16α-OH estrone or the ratio of the two metabolites and breast cancer risk overall.
Including our study, there have been a few reports of significant associations among subgroups of women. However, the specific subgroup is not consistent across studies, nor do the subgroups follow a predicted pattern. For example, we observed a suggestive inverse association with 16α-OH estrone and a significant positive association with the 2:16α-OH estrone ratio among lean women, suggesting possible associations in a low estrogen environment. However, significant associations with both metabolites have been observed in two other studies in environments suggestive of higher estrogen levels, namely high BMI and among women on PMH. Specifically, Modugno et al (33
) observed a combined effect of high BMI and high 16α-OH estrone (RR=3.51, 95% CI (1.34-9.16) for women in the top tertile of BMI and top half of 16α-OH estrone compared with lean women with lower 16α-OH estrone) and Wellejus et al (31
) observed significant positive associations among PMH users with 2-OH estrone (RR for doubling=1.28, 95% CI (1.04-1.56)) and 2:16α-OH estrone ratio (RR for doubling=1.25, 95% CI (1.02-1.53)).
To our knowledge, the study by Wellejus et al (31
) is the only other prospective study to examine these associations by hormone receptor status, although their results were not consistent with ours. In our population of PMH nonusers, we observed no associations with ER+/PR+ tumors, but significant positive associations with 2-OH estrone and the 2:16α-OH estrone ratio among women with ER-/PR- tumors. In the Danish study, no associations were observed with either ER+ or ER- tumors among PMH nonusers but significant positive associations with 2-OH estrone and the 2:16α-OH estrone ratio were observed among PMH users with ER+, but not ER-, tumors. In a retrospective case-control study, Kabat et al (59
) observed a stronger inverse association of the 2:16α-OH estrone ratio with ER- tumors than with ER+ tumors among postmenopausal women. Because circulating estrogen levels have been associated more strongly with ER+/PR+ tumors than with ER-/PR- tumors (2
), it seems contrary that estrogen metabolites may be associated with ER-/PR- tumors. In addition, based on animal studies, 2-OH estrone and the 2:16α-OH estrone ratio have been hypothesized to be inversely associated with breast cancer risk (28
), rather than positively associated as we observed. Given that there are two different, though not necessarily mutually exclusive, hypotheses of the mechanism by which estrogen metabolites may affect breast cancer risk, it is possible that the genotoxicity of 2-OH estrone plays a role in hormone receptor negative tumors (60
). This study has several strengths, including that it is the largest study to date among postmenopausal women not using PMH. Blood samples and risk factor information were collected prior to diagnosis, minimizing the possibility of reverse causality or recall bias. Only one blood sample per woman is a potential limitation, although our reproducibility data suggest that one sample is an adequate representation of these metabolites over at least a few years. Another limitation is the selectivity of estrogen metabolites, with no data on other potentially important metabolites including 4-OH estrone. 4-OH estrogens have a greater estrogenic potential than 2-OH estrogens, given the lower dissociation rate from estrogen receptors compared with estradiol (61
), and are potentially more genotoxic since the quinones form unstable adducts, leading to depurination and mutation in vitro and in vivo (10
). Furthermore, the balance between the catechol (i.e., 2-OH and 4-OH) and methoxy (i.e., 2-Me and 4-Me) estrogens may impact risk. Thus, the investigation of just 2-OH and 16α-OH estrone may be inadequate to rule out the importance of estrogen metabolites on breast cancer risk.
In conclusion, our results do not support the hypothesis that metabolism favoring the 2-OH estrone pathway is more beneficial to breast cancer risk than that favoring the 16α-OH estrone pathway. Though we observed positive associations with 2-OH estrone and the 2:16α-OH estrone ratio among women with lower BMI and women with ER-/PR-tumors, these results were unexpected and require replication. Future studies should include a broader panel of metabolites to investigate the estrogen metabolism pathway and its possible role in breast cancer risk more thoroughly.