BMC Cancer 2008, 8:130 http://www.biomedcentral.com/1471-2407/8/130 water. Slides were then microwaved to retrieve the anti- body-binding epitope of the antigen and treated with casein solution (0.5%) and drained. The slides were then sequentially treated and incubated with the primary anti- body, biotinylated secondary link antibody, and peroxi- dase-conjugated streptavidin. Slides were developed in stable DAB and counterstained with hematoxylin stain. Differences in the procedures at each hospital include that the staining procedures were automated on a Ventana ES system in Kingston but done manually in Toronto, and the primary antibody against the ER, 6F11, was supplied by Ventana in Kingston and by Novocastra in Toronto, and the primary antibody against PR was 1A6 supplied by Ventana in Kingston and 636 supplied by Dako in Toronto. The level of receptors was scored as the percent of immu- nostained epithelial cell nuclei. An experienced patholo- gist with expertise in breast pathology (SKS) assessed sections at 400× magnification under a light microscope. Although immunohistochemical staining expression was examined across the entire slides including all ducts and lobules, only the nonneoplastic tissue without fibrocystic changes on the slides was scored. The percent of cells staining positively for the receptors was scored within cat- egories: < 1, 1–5, 6–10, 11–33, 34–67, and >67. The pathologist was blind to case-control status. Tumor characteristics Levels of ER and PR within the tumors of the cases had been determined by one or both of immunohistochemis- try or enzyme immunoassay in each hospital as part of the routine clinical practice. Using the immunohistochemical assay, tumors were classified as positive if more than 10% of the cells showed nuclear staining for the receptor. Using the enzyme immunoassay, tumors were classified as posi- tive if the concentration of receptor was greater than 10 fmol/mg cytosolic protein. Cases who were positive by at least one assay were considered to have positive receptor status [8]. We obtained the information on ER and PR within the tumors of the cases from the pathology reports. Statistical analyses Because of the differences at each site in the preparation of the nonneoplastic tissue and the immunohistochemical staining, and the differences in the distribution of the ERs by study site that were not explained by case-control sta- tus, menopausal status and age, analyses were also con- ducted separately for each site. Subjects with receptor results were compared to subjects without receptor results with respect to covariates. Among the subjects with expo- sure measurements, frequencies (categorical variables) and means with standard deviations (continuous varia- bles) of the variables were examined. The association between ER and PR content of nonneo- plastic tissue and breast cancer status was examined by calculating odds ratios (ORs) using multivariable logistic regression; this approach is consistent with other studies and allows direct comparison [4,5]. The level of steroid receptors in nonneoplastic tissue was scored in six catego- ries, but adjacent categories were combined such that at least ten cases and ten controls were in each category. Var- iables considered as covariates derived from the question- naire included age (continuous), site (Toronto, Kingston), menopausal status (pre, post), parity (no, yes), lactation duration (continuous), age at first pregnancy (<20, 20– 24, 25–29, > = 30, nulliparous), age at last pregnancy (<25, 25–29, 30–34, > = 35, nulliparous), age last breast fed (never, <30, ≥ 30 years), family history (no, breast can- cer in first or second degree relative), body mass index (continuous), fat intake (continuous), energy intake (con- tinuous), alcohol intake (<1, ≥ 1 drink per week), current smoking (no, yes), cumulative smoking (continuous), and current hormone use (no, yes). Subjects who reported that their menstrual periods had stopped permanently were classified as postmenopausal. However, six subjects who had a hysterectomy in the absence of bilateral oophorectomy and were under the mean age of meno- pause of subjects having a natural menopause (49 years) were reclassified as premenopausal. All ORs were adjusted for age, but no other covariate caused the ORs associated with steroid receptor levels to consistently change more than 10% when introduced into the age-adjusted model and thus, were not included in the models. The confound- ing effect of mammographic density, which had missing values for large numbers of subjects, was examined within the subset of subjects with this measure and was found to be nil. Sensitivity analyses were done to examine if exclusion of cases with carcinomas in situ from the case group, controls with hyperplasia with atypia, subjects who were currently using exogenous hormones, premenopausal women, or subjects with no terminal duct lobular units apparent on the slide had an effect on the conclusions from the main analysis. Polytomous logistic regression was used to esti- mate the association between ER and PR levels in nonne- oplastic tissue and both ER positive breast cancer and ER negative breast cancer relative to the control group. A like- lihood ratio test was performed to evaluate the signifi- cance of the ER and PR levels in nonneoplastic tissue in a logistic model comparing ER positive cases with ER nega- tive cases. The significance of this test indicated if hetero- geneity existed in the associations between ER and PR levels in nonneoplastic tissue and risk of ER-positive and ER-negative breast cancers. Analyses initially were done separately for ER and PR in nonneoplastic tissue, and then they were included in the same model to examine whether they interacted with one another in their associa- Page 3 of 9 (page number not for citation purposes)