BMC Cancer 2008, 8:130 http://www.biomedcentral.com/1471-2407/8/130 Khan et al. [4,5] have found increased ER levels in benign tissue in breast cancer cases than controls even after adjusting for age. In comparison to our unadjusted analy- ses, they observed an increase in risk for ER levels between 1% and 5% relative to less than 1% but no further increases in risk with ER levels beyond 5% [4]. The unad- justed ORs in our study did not increase until higher levels of ER in nonneoplastic tissue were reached. Khan et al. [4] also did not see as strong confounding by age as we did, although in their study the mean age of cases and controls differed by 13 years and age was significantly correlated with ER levels (r = 0.20). In an early study, Ricketts et al. found that mean ER levels determined by enzyme immu- noassay were higher in breast cancer cases than age- matched controls [9]. Another early study found higher levels of ER in tissue taken from a quadrant opposite to a breast lesion in women having mastectomies as compared to tissue taken from reduction mammoplasties, but the ages of these groups were unknown [10]. A nested case- control study done among patients who had benign breast biopsies showing usual ductal hyperplasia found that patients who developed cancer at least six months after this time had nonsignificantly higher median expres- sion of ER in normal tissue and a significantly higher expression of ER in the hyperplastic lesion than age- matched controls who did not develop cancer in the fol- low-up period [11]. A recent study showed that ER levels were nonsignificantly higher in women with fibroadeno- mas than women with either a strong family history with- out known BRCA mutations or a previous history of cancer suggesting that women at high risk of developing breast cancer do not have higher ER expression than women who are not at higher risk of breast cancer [12]. Differences in the methods of determining ER levels in nonneoplastic tissue likely contribute to discrepancies between this study and others, and between the sites in this study. Variation in the distribution of ER in nonneo- plastic tissue is apparent among many studies [11,13-17]. We found differences in the distribution of ER levels between our study sites even when using similarly proc- essed tissue (formalin-fixed, paraffin-embedded), using the same monoclonal antibody (6F11), having a single pathologist to score all the sections, and statistically con- trolling for age, case-control status and menopausal sta- tus. Other factors on which information was not available may have contributed to these differences: a delay in fixa- tion, shorter or longer fixation time, and delay of staining prepared slides are all thought to reduce immunoreactiv- ity with many ER antibodies [18,19]. The distribution of ER was lower in Toronto than Kingston and some degra- dation of the ER possibly occurred at this site. If the distri- bution was shifted downward similarly between cases and controls, a difference should still be seen if there was one. The difference by study site in the distribution of ER levels is a main limitation of this study; it is illustrative of differ- ences that may have occurred between studies but have not been identified because individual studies have been conducted at a single site. If exquisite attention is paid to maintaining strict and unchanging assay methods for all samples within a study, and recording information about parameters (eg. time to fixation, fixation time) that can- not be controlled so adjustment for their effects can be made in the analysis, the observed effect may be stronger with less misclassification and it will be known more clearly if ER degradation occurs differently between cases and controls. The results from most studies are consistent with ER levels in nonneoplastic tissue being increased in women with breast cancer relative to controls. Two interpretations of a difference in steroid hormone receptor levels in nonneoplastic breast tissue between breast cancer cases and benign breast disease controls could be that steroid hormone receptor levels increase the risk of developing breast cancer, or that steroid hormone receptor levels are influenced by adjacent tumors or other pathology. If steroid hormone receptor levels increase the risk of breast cancer, two interrelated mechanisms could be in operation. The first mechanism is that breast tissue with higher expression of ER could be more susceptible to the mitogenic effects of estrogens. Variation in the expres- sion of ER may be due to differences in the level of differ- entiation of the glandular elements in the breast tissue [20], the number of intermediate cells capable of sending mitogenic paracrine signals to the epithelial cells in response to estrogens [21] and/or preneoplastic changes in the breast. Variation in the expression of ERs may have a genetic component but may also be influenced by preg- nancy and lactation history. The second mechanism could involve ER levels being a marker of current estrogen levels; estrogen levels themselves could increase breast cancer risk via ER-dependent stimulation of proliferation or via DNA damage through ER-independent mechanisms by various estrogen metabolites [1,2]. Estrogen receptor lev- els, however, are negatively regulated by current levels of estrogens [3] and were found to be consistently related to only one marker of current estrogens in both sites in this study – age. Analyses that control for current estrogen lev- els measured in blood or breast tissue would help to clar- ify if women whose breast tissues have a tendency to express more ER in the background of a given level of estrogen would be at greater risk for developing a cancer. In this vein, Khan et al. 1999 [22] found that ER levels in nonneoplastic tissue were negatively associated with serum estradiol levels only among benign controls, but not in breast cancer cases. On the other hand, Bhandare et al. 2005 [16] found that estradiol levels in ductal lavage supernatant were not associated with the ER labeling of the epithelial cells in women without cancer (but who did Page 7 of 9 (page number not for citation purposes)