BMC Cancer 2008, 8:130 http://www.biomedcentral.com/1471-2407/8/130 Background Breast cancer risk factors such as early age at menarche, late age at menopause, postmenopausal hormone ther- apy, and high body mass index are thought to affect breast carcinogenesis by increasing the exposure of the breast to estrogens and other sex hormones [1,2]. Estrogens, partic- ularly in conjunction with progesterone, are mitogenic to breast epithelial cells. The resulting proliferation could increase the probability of mistakes being made in DNA replication and setting these mistakes as mutations. Because the effects of estrogens are mediated by the estro- gen receptors (ER), the magnitude of their effects may be determined by the level of ER expressed in the breast. Because breast epithelial cell proliferation is related to both estrogen and progesterone, and progesterone also acts through its own receptor [3], levels of the progester- one receptor (PR) may also be important in breast car- cinogenesis. In previous case-control studies, Khan and colleagues observed that the proportion of breast epithe- lial cells expressing ER was higher in women with breast cancer than benign breast disease controls [4,5]. We previously conducted a hospital-based case-control study of the association between breast cancer risk and breast adipose tissue concentrations of organochlorines such as polychlorinated biphenyls, DDT, and its metabo- lite DDE [6]. Because both breast cancer cases and con- trols had a breast biopsy, we took the opportunity to examine nonneoplastic sections for ERα and PR to see if levels of these receptors differed between the breast cancer cases and controls. Methods The methods of the original biopsy case-control study have been reported in detail elsewhere [6] and the meth- ods specific to this study will be described in detail here. The original and extended protocols were approved by the ethics committees at Women's College Hospital and Queen's University/Kingston General Hospital and all subjects provided informed consent. Subjects Subjects were women who had a diagnostic breast biopsy at Women's College Hospital in Toronto or Kingston Gen- eral Hospital in Kingston between 1995 and 1997. Because the original study depended on obtaining a sam- ple of breast adipose tissue in which to measure organo- chlorines, biopsy controls were chosen. Before biopsy, the study was presented by the surgeons to all women who did not have previous cancer or breast implants and were under the age of 80. After the biopsy, pathology records were reviewed and all women with in situ or invasive breast cancer were cases and all women whose biopsy was negative for malignancy were controls. Of the 824 eligible women, 667 (80.9%) provided informed consent and completed a questionnaire that col- lected information about known and suspected risk fac- tors that were considered as potential confounders in the analyses. Mammographic density was also determined in a subset of 359 women whose mammograms were avail- able to us for analysis. The mammograms were digitized and a computer-assisted thresholding program, Cumulus, allowed separation of the breast area from the back- ground and dense area from nondense area. Percent dense area is calculated by dividing the number of pixels repre- senting dense breast tissue by the total number of pixels in the breast [7]. Immunohistochemistry on nonneoplastic tissue Routine practice at both hospitals from which subjects were recruited was to store surgical specimens from all diagnostic biopsies. We attempted to locate the stored sec- tions of nonneoplastic tissue of the 667 subjects who completed a questionnaire for this study. Stored sections were located for 561 (84.1%) subjects; because this exten- sion study started two years following the time when the last subject had her biopsy and extensive hospital restruc- turing occurred at one of the study sites (Toronto) during this time, some slides were unavailable for review. A sec- tion of nonneoplastic breast tissue from each subject was chosen by a preliminary review of all of the slides stored. The absence of nonneoplastic tissue or unsatisfactory preparation of slides prevented the analysis of the tissue of 36 subjects. Thus, steroid receptor levels in nonneoplastic tissue were determined for 206 cases and 319 controls. Tissue sections were first evaluated by hematoxylin and eosin staining to determine if nonneoplastic epithelium was present to continue with steroid receptor studies. The slides chosen had nonneoplastic tissue including a spec- trum from atrophic, largely fibrous or fatty breast stroma with only scattered ducts and a minor epithelial compo- nent to breast tissues containing large numbers of well formed terminal duct lobular units. In between the two ends of this spectrum was a range of fibrocystic changes, mainly microscopic cysts, lobular unfolding, apocrine metaplasia, and occasional ductal hyperplasia. Rarely the sections of nonneoplastic tissue included a few ducts exhibiting ductal carcinoma in situ, but more frequently, the sections showed a fibroadenoma with a small rim of normal breast tissue around it. Histology apparent on the slide was noted. Steroid receptors were determined using standard immu- nohistochemical techniques; slides at each site were stained no more than two weeks after being cut at the clin- ical pathology departments of the respective hospitals due to feasibility. Briefly, 5 μm sections were treated with 3% hydrogen peroxide solution and washed with distilled Page 2 of 9 (page number not for citation purposes)