This study is the first to show a relationship between the expression of estrogen-dependent genes in ER-positive breast carcinomas and basal levels of estrogen. Although such results may not appear surprising, findings that intratumoral E2 levels are 10 to 20 times higher than those in plasma, and the absence of a reported correlation between levels in the two compartments, have led to the view that intratumoral estrogen synthesis has a greater influence on estrogen signaling than uptake from the circulation.13
The data presented in this article challenge that view and strongly suggest that differences in plasma E2 levels between patients have a significant influence on breast tumors. In particular, this influence may be important to consider in future genomic studies of ER-positive breast cancer, and this may extend to premenopausal women in whom the within-patient changes in E2 levels through the menstrual cycle are marked. Our data relating to Ki67 also suggest that E2 levels merit additional investigation in relation to their possible influence on disease outcome on AIs.
The strength of the relationship between AvERG and plasma E2 levels in this heterogeneous group of ER-positive tumors is remarkable. The relationship was replicated in an independent sample set with good statistical confidence, despite a smaller sample size. The low plasma levels of E2 in postmenopausal women measured in this study would be unquantifiable by most routinely used assays31
and may be one reason that this relationship has not been previously observed. Additional exploration should be conducted only with the use of such specialist assays. In a model system, proportional uptake of E2 from plasma was inversely related to plasma E2 concentrations.32
Our data do not allow us to determine whether this inverse relationship extends to the expression of estrogen-related genes.
The inclusion of ESR1
mRNA expression as a variable along with plasma E2 explained 37% of the variability in the AvERG. There is sufficient unexplained variability for intratumoral synthesis to make a meaningful contribution, but analytic imprecision and other factors also may be influential. One such potential influence examined in this study is the expression of nuclear coregulators.29
Additional exploratory modeling that allowed for an interaction between the five coregulators and ER explained 54% of the variability in AvERG (data not shown). These relationships were not confirmed in the validation set, possibly because of the lower dynamic range on the Agilent platform. These apparent relationships need additional study before they could be considered reliable.
The absence of a relationship between plasma E2 and ESR1
expression and the known effect of estrogen deprivation on the index genes suggest that the relationship between plasma E2 and these estrogen index genes is due to signaling influences rather than to the evolution of particularly estrogen-sensitive tumors as a result of higher E2 levels. As well as providing evidence for the importance of basal levels of E2 on breast cancer biology, the creation of AvERG provides a new index for understanding the clinical and biologic importance of other putative estrogenic influences. For example, a number of steroids thought of largely as androgens also have been found to have significant estrogenic activity in vitro,33,34
and it has been speculated that these may be particularly important in patients under treatment with AIs. They would not be expected to be influenced by such treatment; as a result, they may provide a possible resistance mechanism.33
Assessment of the AvERG in relation to plasma levels of these androgens in AI-treated patients would provide evidence for or against the clinical importance of their estrogenic activity. The AvERG also may be useful as an end point in characterizing the pathologic impact of polymorphic genetic differences in components of the estrogen response mechanism (eg, ESR1
, coregulators) in breast tumors.
Our selection of the four index genes to create the AvERG was based on the well-established estrogen sensitivity of these genes as recorded in numerous publications and, therefore, minimized the potential for false discovery in their selection from tens of thousands of genes. It is likely that, as a result, the current AvERG will not be the most sensitive possible marker of estrogenic activity and that an optimally selected gene set could lead to an improved AvERG.
There are few data assessing the relationship of plasma estrogen levels and outcome of therapy,35
but the weak, significant relationship of baseline plasma E2 level with the value of Ki67 after 2 weeks of aromatase inhibition, a marker we have previously found related to recurrence-free survival,30
suggests that this merits evaluation in large cohorts of patients. The lack of a significant correlation between either plasma E2 or AvERG with clinical response was not surprising given the small patient cohort available. In addition, we have previously found that Ki67 at 2 weeks is a better predictor of long-term outcome on endocrine therapy than clinical response.30
The higher levels of plasma E2 and AvERG in luminal A tumors compared with the other subtypes is consistent with the expression in this group of many ER-related genes.1
Plasma estrogen and androgen levels are significantly correlated with one another. For example, in published data from our laboratory,36
log plasma E2 and log plasma testosterone showed r2
= 0.31 in greater than 2,000 samples from postmenopausal women (correlation data unpublished). This raises the possibility that the correlation between plasma E2 and AvERG could be due in part to plasma testosterone action as a substrate for intratumoral aromatization. Plasma samples from this study, unfortunately, were lost in a fire, which prevented measurements of testosterone to assess this possibility.
In conclusion, this assessment of plasma E2 levels in association with genome-wide expression studies has revealed new relationships that are likely to be important for additional genomic studies of ER-positive breast cancer, for the assessment of multiple putative estrogenic influences on breast cancer, and possibly for clinical outcome after estrogen deprivation therapy.