Growth Factors and Stromal Matrix Proteins Associated with Mammographic Densities Ya-Ping Guo, Lisa J. Martin, Wedad Hanna, Diponkar Banerjee, Naomi Miller, Eve Fishell, Rama Khokha, and Norman F. Boyd 1 Division of Epidemiology and Statistics, Ontario Cancer Institute, Toronto, Ontario M5G 2 M9 [Y-P. G., L. J. M., N. F. B.]; Departments of Pathology [W. H.], and Radiology [E. F.], Sunnybrook and Women’s College Hospital, Toronto, Ontario, M5S 1B2; Department of Pathology, Princess Margaret Hospital, Toronto, Ontario M5G 2 M9 [D. B., N. M., R. K.]; and Division of Experimental Therapeutics, Ontario Cancer Institute, Toronto, Ontario M5G 2 M9 [R. K.], Canada Abstract Extensive radiologically dense breast tissue is associated with a marked increase in breast cancer risk. To explore the biological basis for this association, we have examined the association of growth factors and stromal matrix proteins in breast tissue with mammographic densities. Ninety-two formalin-fixed paraffin blocks of breast tissues surrounding benign lesions were obtained, half from breasts with little or no density and half from breasts with extensive density, matched for age at biopsy. Sections were stained for cell nuclei, total collagen, the stromal matrix regulatory protein tissue metalloproteinase-3 (TIMP-3), and the growth factors, transforming growth factor- and insulin-like growth factor (IGF-I). The area of immunoreactive staining was measured using quantitative microscopy. Breast tissue from subjects with extensive densities had a greater nuclear area (P  0.007), as well as larger stained areas of total collagen (P  0.003), TIMP-3 (P  0.08), and IGF-I (P  0.02) when compared with subjects with little breast density. Differences were greater for subjects less than 50 years of age. These data indicate that increased tissue cellularity, greater amounts of collagen, and increased IGF-I and TIMP-3 expression are found in tissue from mammographically dense breasts and suggest mechanisms that may mediate the associated increased risk of breast cancer. Introduction There is marked variation between individuals in the radiolog- ical appearance of the breast that is attributable to variations in tissue composition. Fat is radiolucent and appears dark on a mammogram, whereas stroma and epithelium are radio-dense and appear light, an appearance that we refer to in this study as mammographic densities (1). Studies that have used quantita- tive methods to classify mammographic densities have consis- tently found that women with dense tissue in more than 60 – 75% of the breast are at four to six times greater risk of breast cancer than women with no density (2). A total of nine studies have been published that have used quantitative methods to classify mammographic densi- ties (2). All of them found significantly elevated odds ratios between extreme categories of density. Although mammo- graphic density is associated with several known risk factors for breast cancer, all of the studies controlled for at least some risk factors for breast cancer, and all of them found that mammographic density was associated with breast cancer risk after adjustment for other risk factors. It has been proposed that the increased risk of breast cancer is an arti- fact, created by the recognized difficulty in detecting cancer in dense tissue. However, in subjects reexamined over an extended period of time, any effects of masking will be short-lived, because cancers missed at one examination will be detected at subsequent examinations (3). Among the eight published studies are two large nested case-control studies performed in cohorts that were screened at annual intervals for several years. Both showed that the increased risk of breast cancer associated with dense breast tissue persisted for extended periods of time, one for at least 5 years and the other for at least 10 years. A more detailed discussion of these issues has been given elsewhere (2). The biological basis for the increase in risk associated with radiologically dense breast tissue is, however, not known. Studies of the histological features of the breast associated with variations in radiological appearance have found that mammographic densities are associated with proliferation of either stroma or epithelium (2), the two types of tissue in the breast with X-ray attenuation characteristics that give rise to radiologically dense breast (1). On the basis of a general model of carcinogenesis proposed by Shigenaga and Ames (4), we propose that the combined effects of two processes, cell pro- liferation (mitogenesis) and damage to the DNA of dividing cells by endogenous mutagens (mutagenesis; Ref. 5), underlie the risk of breast cancer associated with mammographically dense breast tissue. The mammary epithelium, stromal fibroblasts, and myo- epithelial cells communicate by means of several paracrine signals (6 – 8), and we hypothesize that growth factors within the stroma influence stromal cell proliferation and matrix dep- osition, which contribute to mammographically dense breast tissue and either directly or indirectly affect the epithelium. In this study, we have examined the quantity of cell nuclei, col- lagen, and selected molecular factors in breast tissue in relation to mammographic densities. Compared with tissue from breasts with little radiological density, we found significantly greater Received 6/9/00; revised 12/14/00; accepted 12/26/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Division of Epidemiology and Statistics, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2 M9, Canada. Phone: (416) 946-2945; Fax (416) 946-2024; E-mail: boyd@oci.utoronto.ca. 243 Vol. 10, 243–248, March 2001 Cancer Epidemiology, Biomarkers & Prevention