Introduction
Assessment of the risk of development of invasive breast cancer has recently become a significant problem. This is partly due to the recognition that invasive breast cancer can be prevented by mammographic detection and the treatment of earlier pre-invasive breast lesions [
1,
2]. These lesions have been defined by epidemiological, histological and molecular observations and comprise progressive morphological changes in breast epithelium that might parallel the process of evolution towards invasive carcinoma [
3,
4].
Mammographic density is a risk factor for breast cancer and is attributed to alterations in the composition of breast tissue [
5,
6]. Previous studies seeking to understand the biological basis of mammographic density have focused on associations with epithelial changes [
7‐
10]. However, the major tissue component in breast is stroma, and stromal alterations are also a well recognized component of benign and pre-invasive breast lesions. Furthermore, although breast cancer is a direct manifestation of alterations in the expression of multiple genes and cellular pathways within the breast epithelial cell, it is now recognized that perturbations in stromal–epithelial interactions might also influence tumorigenesis and progression through direct effects on growth factor pathways and indirect effects mediated through cell adhesion and structure [
9‐
12].
We and others have previously shown that distinct genetic alterations can occur in the stromal compartment of breast tumours [
13‐
15]. We have also shown that the small leucine-rich proteoglycans (SLRPs) lumican and decorin are a highly abundant component of breast tissue stroma and that altered expression of stromal proteins is associated with tumour progression and outcome [
16,
17]. These SLRPs might be important in both stromal integrity and growth factor pathways through their influence on fibrillary collagen cross linking and, at least for decorin, the ability to bind to growth factors such as transforming growth factor-β and growth factor receptors such as epidermal growth factor [
18‐
21].
We therefore wished to examine the relationship between mammographic density, breast tissue composition and expression of stromal proteins, to establish whether the increased risk attributed to increased mammographic density might reflect stromal alteration.
Discussion
Mammographic density might be a risk factor for the development of breast cancer [
5,
6]. An improved understanding of factors influencing changes in mammographic density would improve its value and practical application in risk assessment. Mammographic density has been shown to be influenced by age, menopausal status and dietary factors and also by inherited genetic factors [
9,
10,
23‐
34]. However, the biological basis underlying variations in mammographic density is unknown [
35].
Many studies to explore these factors have examined the relationship between mammographic density and breast tissue pathology [
8‐
10,
31,
32,
36,
37]. Although several of these have also noted a close association with stromal changes [
32,
36‐
38], the tendency to focus principally on the relationship between increased mammographic density and specific epithelial lesions persists [
7,
10,
31,
39]. The notion that increased mammographic density is attributable to the presence of more 'glandular tissue' reflects this view [
35]. However, although statistically significant, the strength of the correlation between mammographic density and proliferative lesions in most studies is relatively weak.
The major tissue fraction in breast is stroma, not epithelial tissue. An alternative explanation is therefore that changes in mammographic density primarily reflect stromal changes and that these changes influence breast cancer risk [
32]. Alteration of the stromal architecture and composition of the extracellular matrix is a well-recognized component of both benign and malignant breast pathologies, from the fibrosis within low-risk benign non-proliferative lesions (often encompassed by the term 'fibrocystic changes') through typical and atypical proliferative ductal lesions, to the 'stromal reaction' associated with ductal
in situ and invasive carcinoma. Although the 'host reaction' in the latter malignant lesions has been attributed to paracrine perturbations originating from the epithelial tumour cell, fibrosis and stromal changes also occur in the absence of epithelial proliferation and can precede it. Recent studies have shown that both stromal architecture and composition can exert an important influence on normal epithelial biology [
11,
40], and somatic mutations can be identified in the stromal compartment of breast tumours independently of mutations in the neoplastic epithelium [
13,
14]. These observations are in keeping with the concept that stromal alterations might not always be 'reactive' but might sometimes play an initial 'landscaping' role in breast carcinogenesis, as has been proposed for the colon [
41,
42].
In the present study we have also found a significant association between increased mammographic density and high-risk DCIS lesions. However, despite agreement with several previous studies, some limitations to the broader interpretation of this study should be noted. The study cohort was biased towards patients undergoing biopsies after the identification of lesions by screening mammography, and the majority of women were also probably postmenopausal, reducing the proportion of cases in the category of high mammographic density [
25]. The mammographic density was assessed by subjective categorization, although we used well-established criteria applied to mammograms performed within a standardized screening programme and restricted our study set to patients with clearly distinguishable extents of mammographic density (less than 25% or more than 50% of the mammogram occupied by dense tissue). The relationship between the region assessed for density and the tissue blocks examined was not precise. However, we restricted our assessments of mammographic density and corresponding tissue to the margins of region of tissue encompassed by the subsequent biopsy, ignoring the locations influenced by the detected lesion.
Although there was an association with the occurrence of DCIS, our study showed no significant correlation between mammographic density and epithelial proliferation or density of epithelial duct-lobular units within the corresponding region of breast tissue beyond the primary lesion. Instead, there was a close correlation between the extent of collagenous stroma measured with several indices and its composition measured by the expression of the stromal proteoglycans lumican and decorin.
Breast stroma includes a variety of extracellular matrix proteins, of which the fibrillar collagens are perhaps the most important in determining stromal architecture. These are secreted as triple-helical procollagen molecules that undergo extracellular processing and assembly into collagen fibrils followed by cross-linking and aggregation to form collagen fibres. Fibrillogenesis and fibril spacing are important aspects of stromal integrity and are affected by several structural proteins and by proteoglycans, including the SLRPs lumican and decorin [
18,
20], which are highly abundant in the stroma [
16,
43]. Decorin can also exert biological effects directly through its influence on growth factors and growth factor receptors [
19,
21]. The levels of expression of these SLRPs are correlated when compared within non-neoplastic or within neoplastic tissues, and low levels of both SLRPs are associated with poor outcome in primary invasive tumours. This latter observation might reflect a differential host response to tumour epithelium [
17], although SLRP expression can vary independently of epithelial proliferative changes, as shown here, and alteration in the lumican : decorin ratio from that in normal tissue adjacent to the invasive margin can occur in neoplastic stroma [
16]. The principal factors determining SLRP expression in breast are not known, although the expression of insulin-like growth factor (IGF) has been implicated as a risk factor for breast cancer [
44] and is also known to be associated with mammographic density in premenopausal women [
45]. IGF-1 can also induce lumican, decorin and collagen synthesis in model systems [
46‐
48], with most influence on cells from younger patients [
48], and both lumican and IGF-1 are often induced in benign disease in other tissues [
49]. These last observations are consistent with the hypothesis that increased mammographic density might reflect primary stromal alterations, including SLRP expression, that could influence early tumorigenesis.