BMC Cancer 2009, 9:182 http://www.biomedcentral.com/1471-2407/9/182 improve our understanding of the relation between den- sity and breast cancer risk as well as the potential for risk prediction and modification. The two types of tissue that give rise to radiologically dense breasts are epithelium and stroma forming the microenvironment of epithelial cells which constitute less than 5% of breast tissue [3,4]. The main component of stromal tissue is collagen [5]. It was hypothesized that the extent of mammographic density is proportional to the amount of breast epithelium and that the higher breast cancer risk associated with breast density is due to a larger number of glandular cells at risk for malignant transformation [6,7]. This idea is supported by findings of an association between the proliferation of stroma, epithelium, or both with breast density in subjects with breast abnormalities [8,9]. Unfortunately, research in healthy women is limited to forensic studies [10,11] and one study of breast reduction samples [5]. In breast cancer patients, increased amounts of collagen were asso- ciated with breast density in several reports [5,9,11,12], while the results on cell proliferation were mixed [5,13,14]. As risk factors that induce cell proliferation [15,16], endogenous sex steroids and hormone therapy (HT) are associated with higher breast cancer risk [17,18]. Whereas HT, in particular estrogen plus progestogen therapy, increases mammographic density [19], the relation between endogenous sex steroids and mammographic density is less clear. One study observed an association with endogenous estrogens [20] but others did not [21- 23]. As breast tissue levels are partly determined by estro- gen production in adipose tissue, breast size as marker for adipose tissue surrounding the epithelial cells may possi- bly be a better marker for tissue levels than circulating estrogen levels. Endogenous progesterone was found to be related to mammographic density in one report among premenopausal women [24]. The biological activities of endogenous and exogenous estrogens on breast tissue are mediated by nuclear estrogen receptors (ER) α and β. Dif- ferential effects of ERα and ERβ are of interest because ERβ appears to be more antiproliferative while ERα has prolif- erative activity [25,26]. Progesterone, an ovarian steroidal hormone, acts through its specific receptor (PR) [27,28]; PR expression has been shown to be a sensitive indicator of estrogenic effects in cells [29]. To understand how hormone receptors and cell prolifera- tion are related to breast density, this study examined the expression of ERα, ERβ, and PR as well as HER2, Ki-67, and Proliferating Cell Nuclear Antigen (PCNA) [30], in relation to mammographic density among breast cancer patients with Caucasian, Japanese, and Hawaiian ethnic- ity. We are convinced that these associations are best stud- ied in benign breast tissue and, thus, restricted this analysis to breast cancer patients for whom benign tissue samples placed on tissue microarrays (TMA) were availa- ble. The relation between mammographic appearance of the breast and marker expression in tumor tissue is a sep- arate issue that needs further study [31]. Methods Study population The study was approved by the Institutional Review Boards of the University of Hawaii and Wake Forest Uni- versity; all subjects provided informed consent in writing. We recruited subjects for the TMA study from 607 breast cancer cases who had participated in the Multiethnic Cohort (MEC) [32] and a nested case-control (NCC) study of mammographic densities [33]. Of these, 177 women were excluded because their tumor blocks were not available from the Hawaii Tumor Registry (HTR). Recruitment letters and questionnaires were mailed to the remaining 430 subjects; 323 (75%) women returned the consent forms. Another 12 women were deceased but linked to the HTR and could, thus, be included in the study. For 279 out of these 335 subjects, pathologic blocks from breast cancer surgery were located and used to create TMAs; no tissue from prior benign biopsies was available. At entry into the MEC, all participants had completed a questionnaire that inquired about demographics, repro- ductive behavior, anthropometric measures, and family history of breast cancer [32]. As part of the NCC, women completed an additional one-page breast health question- naire that asked about previous breast surgery, menopau- sal status, mammography history, and HT use [33]. Tumor microarrays TMAs were prepared according to standard procedures [34,35]. In brief, a surgical pathologist (JK) identified blocks from a given patient with sufficient tissue. For each of these blocks a single hematoxylin and eosin (H&E) slide was prepared on which the same pathologist marked representative areas of malignant and benign tissue. The H&E slide was aligned with the corresponding "donor" block and a 0.6 mm cylindrical tissue specimen was taken from the selected area and transferred to a "recipient" par- affin block using a tissue-arraying instrument (Beecher Instruments, Sun Prairie, WI). When available, four malig- nant cores and four benign cores per patient were placed in one of six blocks. Out of the 2,232 cores to be placed (four malignant and four benign samples for 279 women), tissue was insufficient for 12% of malignant and 29% of benign specimens resulting in 1,773 tissue sam- ples (79.4%) for analysis. At least one benign or malig- nant core was available for 268 women. Several 5 μm sections were mailed to Wake Forest University for immu- nohistochemical staining. Immunohistochemistry and pathologic evaluation The TMAs were stained for the following markers: ERα, ERβ, PR, and PCNA (Clones 6F11, EMR02, 1A6, and PC10, respectively; all from Novocastra Labs, Newcastle- Page 2 of 8 (page number not for citation purposes)