Cellcell adhesion

Epithelial cells mediate intercellular adhesion primarily through adherens junctions, desmo-somes, and gap junctions. The molecules involved in these adhesive complexes - classical cadherins, desmosomal glycoproteins and connexins, respectively - have all been shown to exhibit altered expression in breast cancers.

The classical cadherins include E-cadherin, P-cadherin and N-cadherin, and of these the major focus in breast cancer has been E-cad-herin. E-cadherin mediates homophilic Ca++-dependent adhesion and, via interactions with cytoplasmic catenins and the actin cytoskeleton, it plays an important role in maintaining epithelial morphogenesis.1 There is compelling evidence to indicate that E-cadherin acts as a tumor suppressor: in vitro studies have indicated an invasion-suppressor role for E-cadherin,2,3 whilst loss of heterozygosity (LOH) at this site is frequently detected in breast carcinomas,4 and hypermethylation of the E-cadherin promoter region, with reduced expression of the protein, is also common in breast cancer.5,6 Numerous studies have examined the relationship between downregulated E-cadherin and breast cancer behavior. In infiltrating ductal carcinoma (IDC), reduced membrane E-cadherin has been associated with high tumor grade7-9 and the presence of lymph node metastases,9,10 though other studies show no relationship with conventional prognostic indices.11,12 However, a recent meta-analysis confirmed interstudy heterogeneity, but an analysis of 10 retrospective studies found that reduced or absent E-cad-herin significantly increased the risk of all-cause mortality, whilst a nonspecific association was identified for breast cancer-specific mortality.13 In addition to reflecting differences in study design, the contradictory findings regarding the prognostic value of E-cadherin also likely reflects the complexity of its role in breast cancer spread. Thus, it has been suggested that downregulation of E-cadherin may be a transient event, with reexpression at a distant site;14 and enhanced E-cadherin expression in nodal metastasis has been shown to be an independent marker of improved survival, whilst no such relationship was shown with E-cadherin levels in the primary tumor.15

In contrast to the tumor-suppressor function of E-cadherin, expression of P-cadherin in invasive breast carcinomas is consistently associated with features of more aggressive tumors, including high tumor grade and estrogen receptor negativity16,17 the presence of lymph node metastasis,18 and reduced disease-free and overall survival.17 Recent gene expression array studies cluster P-cadherin expression with the basal subtype of breast cancer,19 which is also associated with poor patient outcome.20 Furthermore, P-cadherin expression is strongly associated with BRCA-1-mutated breast carcinomas, and has been shown to be a predictor of poor prognosis, particularly in small node-negative tumors.21 Expression of P-cadherin appears to be controlled primarily through methylation;17,22 whilst the function of P-cadherin is poorly understood, one study demonstrated a pro-invasive effect of this cell adhesion molecule.23

Altered expression of other members of the cadherin family has also been reported in breast cancer, though little is known of their functional and prognostic impact. N-cadherin has been detected in up to 30% of invasive breast cancers,24 and has been shown to promote tumor cell invasion25 and enhance tumor metastasis in animal models.26 However, limited studies to date suggest expression in primary breast carcinoma does not relate to tumor prognosis.24

Desmosomes are another major adhesive complex in epithelial cells, of which the desmosomal glycoprotein families of desmo-collins (DSc) and desmogleins (DSg) are key components.27,28 DSc and DSg exhibit a tissue-specific pattern of expression; in the breast DSc1 and -2 and DSg 1 and -2 proteins are expressed by all epithelial cells, whilst DSc3 and DSg3 are restricted to myoepithelial cells in the normal breast.29 In the normal breast, desmosomal proteins mediate cell adhesion, induce polarity of mammary epithelium29 and inhibit cell motility.30 Despite a central role in maintaining tissue structure, little is understood about changes in desmosomes in cancer. Downregulation of DSc3 has been described as a common event in breast can-cer,31,32 which is frequently associated with promoter methylation.32 Other desmosomal components are also downregulated in tumor compared to normal tissues, including desmo-plakin and desmoglein 2,33 though the functional importance of such changes remains to be established.

The connexin family of gap junction proteins are involved in regulation of cell growth, cell differentiation and tissue development, and they are widely regarded as having a tumor-suppressor role.34,35 Connexin 26 (Cx26) and Cx43 are expressed in normal breast epithelium, and both reduced and enhanced levels of expression in breast carcinomas have been reported.36,37 Experimental systems have indicated a dominant role for these proteins in control of breast differentiation, with overexpression of Cx26 and/or Cx43 leading to the reversion of the malignant phenotype through regulation of epithelial-mesenchymal transition and angiogenesis.38 Once again, despite powerful experimental

Figure 5.1 Integrin heterodiners. Each integrin comprises an a and P subchain which combine to form 23 integrins. Note that a4 combines with both P1 and P7, and a6 combines with P1 and P4.

Figure 5.1 Integrin heterodiners. Each integrin comprises an a and P subchain which combine to form 23 integrins. Note that a4 combines with both P1 and P7, and a6 combines with P1 and P4.

evidence, the predictive value of assessing con-nexin expression in primary breast cancer has not been fully evaluated. It may not be as straightforward as anticipated, since there are suggestions that the functional impact of con-nexin expression may be context dependant, and there may be instances where they contribute to, rather than suppress, breast cancer

progression.39 Cell-matrix adhesion

The integrin family of cell adhesion molecules is the major mediator of cellular interaction with the extracellular matrix. Integrins are cell surface receptors composed of non-covalently linked a and P subunits,40 and at least 22 heterodimer receptors are now recognized (Figure 5.1). Cell-matrix interactions mediate many of the processes implicated in tumorigenesis, including proliferation, differentiation, migration and invasion,41 thus, changes in expression on breast carcinoma cells may be expected to have an important impact on tumor cell behavior.

The integrin receptor profile for normal breast epithelium includes a2pi, a3pi, a6pi and a6P4, with low level expression of a5pi and avP3.42,43 Strong expression of many of these receptors is localized, particularly within the myoepithelial compartment, and a6P4 is largely confined to the junction with the basement membrane, consistent with its incorporation into hemidesmosomes. Evidence for the role of integrins in modulating tumor cell behavior comes both from in vitro functional studies, and tissue studies, though there are some discrepancies between these approaches. Thus, in three-dimensional culture models of breast cancer, blocking pi integrin leads to increased apoptosis and decreased prolifera-tion,44 and induction of normal breast morphogenesis,45 suggesting that pi integrin promotes tumorigenesis. However, in apparent contradiction to this, a number of tissue studies report that reduced levels of pi integrin are associated with higher tumor grade and with axillary lymph node metastases.43,45-47 The simplest explanation for this discrepancy is that tissue studies do not measure levels of activated integrin, which is likely to be important. In support of this, activated (but not nonactivated) avp3 integrin has been implicated in promoting tumor metastasis.48 However, a recent tissue study has found that high level pi integrin staining on breast carcinomas is an independent predictor of disease-free and overall survival.49 Whilst this appears more in keeping with the in vitro findings, it is difficult to reconcile with earlier studies, though it is notable that the latter study is the largest of all those reported, which is probably a result of successful antigen retrieval techniques allowing application to routinely fixed archival tissues. The possibility that pi integrin could be targeted for ther-apy49-51 means that further large-scale studies to determine the prognostic role of pi inte-grin are imperative.

Another integrin heterodimer which plays a complex role in breast cancer is a6p4 integrin. In the normal breast, a6p4 is largely confined to the cell-basement membrane interface where it is incorporated into hemidesmosomes. Reduced or absent a6P4 has been a consistent finding in many studies of primary breast can-cer,464852 though when detected it has been associated with poor patient prognosis in some series53 but not in others.54 Methodological

\ps differences between these studies may explain the discrepant findings. And both studies have limitations: the former is on a small sample cohort; the latter depends on in situ hybridization, which may not reflect the level of protein expression. Thus, the true prognostic significance of a6P4 in breast cancer is not fully established, though molecular subtyping using gene expression arrays has identified a6P4 integrin as one of the genes associated with the basal subtype of breast cancer,19 and it may well contribute to the aggressive nature characteristic of this tumor group.20 Certainly, experimental data indicate a role for a6P4 integrin in promoting tumor cell growth and invasion,55-57 mediated at least in part through its collaboration with other signalling molecules such as c-met58 and c-erbB2.59 However, other data suggest an antitumor effect of a6P4 integrin: Weaver et al60 found that upregulation of a6P4 in breast cancer cells reversed some features of the malignant phenotype and promoted glandular morphogenesis. Furthermore, upregulation of a6P4 has been shown to restore contact inhibition of growth61 and reduce breast cancer cell invasion.62 These apparent contradictions perhaps result from the different functions of a6P4 depending on its cytoskeletal attachments, as it mediates anchorage through intermediate filament-associated hemidesmo-somes but migration in actin-associated adhesive structures.63,64 Furthermore, the effect of a6p4 signalling may be dependent on coex-pression of other molecules such as c-met. Such relationships require further investigation in primary tissues in order to fully understand the prognostic significance of a6p4 integrin.

The integrin avp6 is of interest in that it is epithelial specific; it is expressed weakly or is absent on normal adult epithelia, but is increased in injured or inflamed epithelium.65,66 Importantly, high expression of avP6 integrin has been detected on many cancers67 and correlates with reduced survival from colon cancer.68 Work in our group has shown that upregulation of avp6 integrin is significantly associated with high tumor grade, though not lymph node status, but is an independent predictor of poor patient outcome (unpublished data). Since avP6 is rarely expressed in normal tissue, and has been shown to promote tumor cell invasion, it presents a plausible target for therapeutic attack.

STROMAL CHANGES IN BREAST CANCER

It has long been recognized that the stroma associated with breast carcinomas differs from normal;69 however, it is only in the last decade that the critical role of the microenvironment in determining tumor behavior has been acknowledged. Indeed, a number of in vivo model studies suggest that stromal alterations alone can lead to induction of mammary car-cinoma.70,71 There are many components to the stromal microenvironment, each of which can contribute to the modulation of tumor behavior. Key features include cellular components, such as fibroblasts and inflammatory cells, and the extracellular matrix proteins. The tumor-associated vasculature clearly has an important influence on tumor behavior but is not covered here since this is a topic in its own right.

Cellular changes in the breast cancer microenvironment

One of the main cellular components of the stroma is the fibroblast population, which undergoes activation in the tumor environment to form myofibroblasts72 which have a pleiotropic effect on tumor cells and the environment. Differences in the pattern of gene and protein expression have been identified between peri-tumoral and normal fibroblasts,73,74 and a number of these gene families are implicated in promotion of tumor growth and invasion. For example, it has recently been shown that tumor-associated fibroblasts (TAFs) secrete high levels of the chemokine stromal cell-derived factor-1 (SDF-1), which binds to the CXCR4 receptor on breast cancer cells, promotes tumor growth and invasion,75 and is critical for metastatic spread of breast cancer cells to bone and lung.76 In keeping with this experimental data, it has been shown that elevated SDF-1 levels in human breast carcinomas correlate with the presence of lymph node metastases, and with reduced disease-free and overall survival.77

Fibroblasts are also the major source of extracellular matrix proteins and matrix-degrading proteolytic enzymes, both of which have important functions in tumor progression, and are discussed further below. However, as the contribution of TAFs to breast cancer progression becomes more widely accepted, attention has focused on determining the precise nature of these cells and how their altered function is generated. The changes in gene expression exhibited by TAFs are thought to arise largely as a response to tumor-derived signals; however, there is growing evidence to indicate that the peri-tumoral stromal population may undergo independent genetic and epigenetic modifications,78-81 and such changes may influence the function of the stromal population and contribute to their tumor-promoter role. It has also been suggested that independently acquired genetic alterations in the stromal population may influence the diversity in clinical outcome observed in breast cancer.78 This has recently been illustrated in an analysis of p53 mutations in tumor-associated stroma, whereby somatic p53 mutations in the stroma (but not in the epithelium) of breast cancer were associated with regional lymph node metastases,82 and in the absence of p53 mutations, loss of heterozygosity and allelic imbalance at other loci in the stroma associated with metastases. This is one of the first studies to provide definitive evidence of the importance of stromal changes in breast cancer.

Changes in the extracellular matrix in breast cancer

The extracellular matrix (ECM) provides a scaffold for epithelial cells in tissues; through direct adhesive interactions, or via cross-talk with classical signalling cascades, the ECM has a central role in controlling epithelial cell growth, differentiation and migration.83-85

Basement membrane (BM) represents a specialized form of the ECM laid down at epithelial-stromal junctions and around blood vessels. In addition to a modulatory role, ECM and BM act as important physical barriers to invasion by tumor cells. It is clear then that changes in the composition and integrity of the ECM may profoundly influence tumor behavior.

The ECM around breast cancer differs from normal breast.86 Fibronectin expression is increased in the stroma of many breast cancers, and changes in the isoform profile have been described with upregulation of protein containing the so-called ED-A and ED-B domains.87,88 Whilst many in vitro studies indicate a role for fibronectin, particularly in promoting breast cancer cell motility or growth,89,90 few tissue studies have established the prognostic value of enhanced fibronectin expression. Yao et al49 recently demonstrated that high fibronectin expression was associated with reduced disease-free and overall survival in univariate, but not multivariate, analysis. This finding was in agreement with an earlier immunohistochemical study,91 though this conclusion has not been universal.92 Interestingly, most studies do not distinguish between the different fibronectin isoforms, which is likely to have an important influence on the results. A distinct form of truncated fibronectin, termed migration-stimulating factor (MSF), has been characterized; as the name implies, MSF stimulates cell migration but expression is confined to fetal and tumor tissues.93 However, the prognostic significance of this isoform has not yet been established.

Another ECM protein shown to be consistently upregulated in breast cancer is Tenascin-C (TN-C). TN-C is a multifunctional protein which can influence cell behavior directly through interactions with cell surface receptors, and indirectly through binding to other matrix proteins such as fibronectin, and altering their interaction with cells.94 High expression of TN-C has been related to the presence of lymph node metastases,91 local and distant recurrence,95 and reduced sur-vival;91 expression of TN-C in DCIS has been suggested to predict progression to invasion.96 However, as with many of the ECM proteins, diversity is generated through expression of alternatively spliced isoforms, which introduces functionally relevant domains into the mature protein.97 Several studies have indicated a switch towards larger molecular weight isoforms in tumor tissues compared to normal.98-100 Work in our laboratory has detected very specific changes in TN-C isoform profile in breast cancers, with induction of two isoforms not usually found in normal breast tissue: one containing exon 16 (TN-C16) and one containing exons 14 plus 16 (TN-C14/16).101 Whilst these isoforms appear to specifically promote breast cancer growth and invasion (unpublished data), their prognostic value is not yet established, partly due to lack of good reagents to these splice variants. A further member of the Tenascin family is TN-W, which has recently been shown to be upregulated in breast cancers though is undetectable in normal breast tissue.102 Interestingly, TN-W appears to be particularly upregulated in low-grade breast cancers, and has been suggested to be an early marker of activated tumor stroma.102 Extending the work on Tenascin members and their isoforms is likely to prove valuable since tumor-specific TN-C isoforms are already being successfully targeted in other malignancies.103

Further emphasizing the importance of tumor-specific splice variants, a recent report has shown that a switch in laminin isoform profile, from p2-containing to pi-containing laminins, occurs during progression of breast cancer.104 These novel laminin isoforms are deposited in newly formed tumor blood vessels and again represent a potential therapeutic target.

From this discussion it is very apparent that the changes in ECM in tumors are complex. A recent study used gene expression microarray analysis to determine the patterns of ECM changes in breast cancer.105 They showed that breast cancers could be classified according to their profile of ECM expression, and that this had clinical significance with tumors exhibiting overexpression of protease inhibitors having a favorable outcome, whilst those with high expression of integrin and metallopepti-dases having a poor prognosis.105

Matrix remodeling in breast cancer

In addition to changes in the extracellular matrix composition, the matrix is also altered through remodeling. This is generated through the action of proteolytic enzymes, of which there are many but those most commonly implicated in cancer include the matrix metalloproteinases (MMPs), the uroki-nase plasminogen activator system and the A disintegrin and metalloproteinases (ADAMs). Each of these protein families has been shown through, in vivo and in vitro model systems, to play a role in cancer progression, and parallel tissue studies are starting to identify their potential prognostic value. A large body of literature surrounds the MMP family and accordingly this discussion will focus on the role of the MMPs in breast cancer.

Matrix metalloproteinases

The human MMP family comprises 24 members which between them can degrade virtually all components of the extracellular matrix.106-108 They are zinc-binding endopep-tidases which share a number of functional domains including: (i) a signal peptide required for secretion; (ii) a propeptide domain which interacts with the zinc-binding site and maintains the enzyme in an inactive form; (iii) a catalytic domain which contains the zinc-binding site with the exception of the matrilysins; and (iv) a hemopexin/vitronectin-like domain connected to the catalytic domain via a hinge. Traditionally, MMPs have been classified according to their substrate specificity but with the growing complexity of the family, and their overlapping activity, they are increasingly classified on a structural basis (see Figure 5.2).

One of the defining characteristics of MMPs is the tight regulation of their activity. They are under the control of a variety of naturally occurring inhibitors including the

Enzyme

Substrate

Structure

Matrilysin (MMP-7) Matrilysin 2 (MMP-26) Collagenase 1 (MMP-1) Collagenase 2 (MMP-8) Collagenase 3 (MMP-13)

Stromelysin 1 (MMP-3)

Stromelysin 2 (MMP-10)

Metalloelastase (MMP-12) RASI (MMP-19) Enamelysin (MMP-20)

MMP-27, C-MMP (MMP-22) Stomelysin 3 (MMP-11) X-MMP (MMP-21) Epilysin (MMP-28) Gelatinase A (MMP-2)

Gelatinase B (MMP-9)

MT 1-MMP (MMP-14) MT 2-MMP (MMP-15) MT 3-MMP (MMP-16) MT 5-MMP (MMP-24) MT 4-MMP (MMP-17) MT 6-MMP (MMP-25)

Proteoglycan core protein FN, Ln, denatured collagens

Denatured collagens Collagens I, II, III, VII, X Collagens I, II, III

Collagens I, II, denatured collagens, aggrecan

Proteoglycan core protein FN, Ln, denatured collagens Collagens IV, V, IX, X

Denatured collagens Collagens III, IV, V

Stromelysin-like Ameloganin

Not established

Denatured collagens

Denatured collagens, Native collagens IV, V VII, X, FN, Elastin

Denatured collagens, Native collagens IV, V

Pro-MMP-2, pro-MMP-13, FN, Nidogen, aggregan, collagen I, III Pro-MMP-2, Ln

Pro-MMP-2 TNFa convertase Pro-MMP-2 Not established

rHBH

rllB

rHBH

I Predomain ^J Signal anchor

Pro-domain JJ Furine domain

Catalytic domain • Zinc-binding site

Transmembrane domain GPI anchor domain

Hemopexin domain B Cytop|asm|c domain Hinge domain

Cysteine array Ig-like domain

Elastin

Figure 5.2 Classification and structure of matrix metalloproteinases (MMPs). MMPs may be classified according to substrate specificity (collagenases, stromelysins, gelatinases) or according to structural similarity. The simplest MMP is the Matrilysin subgroup, comprising a signal prepreptide domain, a propeptide that maintains the enzyme in the inactive form, and the catalytic domain with the zinc-binding site. The collagenases, stromelysins, metalloelastase, enamelysin, MMP-19 and MMP-27 contain an additional hemopexin domain, which provides substrate specificity. The gelatinases also contain a series of fibronectin type II units, whilst stromelysin-3, epilysin and MMP-21 have a furin-like cleavage site which allows intracellular activation. The membrane type (MT)-MMPs form a distinct group and are linked to the cell membrane either via a transmembrane domain or with a glycosylophosphatidyl-inositol (GPI) anchor. CA-MMP contains a unique cystein array and immunoglobulin-like domain in the C-terminal, with an N-terminal signal anchor targeting it to the cell membrane. (Adapted from Chabottaux and Noel.108)

tissue inhibitors of metalloproteinases (TIMPs) of which there are four,109-112 as well as the plasma inhibitor a2-macroglobulin113 and the so-called reversion-inducing cysteine-rich protein with Kazal motifs (RECK).114 The majority of MMPs are secreted as inactive precursors or may be secreted in their active form following cleavage of the propeptide intracellularly by furin-like convertases.115 A distinct group of MMPs are membrane associated, either via a transmembrane domain (MT1-, MT2-, MT3-and MT5-MMP), a glycosylphosphatidyl-inositol (GPI) anchor (MT4- and MT6-MMP) or an N-terminal signal anchor (SA) targeting it to the membrane (CA-MMP).116

In addition to their classical role in matrix degradation, MMPs are becoming recognized for a much broader range of activities. Thus, MMPs can control cell proliferation through release of matrix-bound growth factors, or activation of latent growth factors, such as MMP-3 release of insulin-like growth factor117 or MMP-7 activation of heparin-binding epidermal growth factor (EGF)-like growth factor.118 Both MMP-1 and MMP-3 have been shown to break down perlecan leading to the release of basic fibroblast growth factor (FGF), which is a potent mitogen for endothelial cells.119 A number of other MMPs are involved in angiogenesis, including the gelati-nases120 or some of the MT-MMPs which can activate vascular endothelial growth factor (VEGF)121 or directly enhance vascular tubu-logenesis.122 In contrast to the pro-angiogenic role of most MMPs, MMP-19 appears to be a negative regulator of tumor angiogenesis.123 Many of the MMPs are also involved in mediating tumor cell invasion. The MT-MMPs have been implicated in directly breaching basement membrane barriers through the assembly of invasive pseudopodia.124 MMP-3 and MMP-7 have been shown to enhance tumor invasion through cleavage of E-cadherin and induction of the epithelial-mesenchymal transition (EMT).125,126 Having such multifac-eted roles in the processes relevant to tumor progression, the relationship between MMP expression and prognosis has been much studied in breast cancer, and despite the complexity some clear patterns are beginning to emerge.

A number of studies have demonstrated a relationship between elevated gelatinase levels and unfavorable prognosis in breast cancer. Iwata et al127 reported significantly higher levels of MMP-2 in lymph node-positive breast cancers compared to lymph node-negative ones, and elevated MMP-2 and MMP-9 relative to their inhibitors TIMP-2 and TIMP-1 have been associated with lymph node positivity and reduced survival.128,129 In a separate study, MMP-2 positivity in breast cancer was identified as an independent predictor of reduced disease-free and overall survival,130 and has also been shown to predict poor response to antiestrogen therapy.131 The prognostic value of MMP-9 is less consistent, with some studies reporting a positive association with more aggressive disease,132 others no associ-ation133,134 and even an inverse relationship with outcome; Scorilas et al135 found overexpression of MMP-9 to be an independent predictor of improved survival in node-negative patients. In contrast, two recent studies have shown MMP-9 to be related to reduced sur-vival136 and to act as an independent predictor of poor prognosis.137

The MT-MMPs are emerging as key enzymes in promoting breast cancer progres-sion.124,138 Several studies have demonstrated a relationship between MT1-MMP and the presence of lymph node and/or distant metastases:133139140 and elevated MT1-MMP mRNA in breast carcinomas has been shown to predict significantly reduced survival even when adjusted for factors such as tumor size and lymph node status.141 No such correlation has been shown with MT2-MMP, and MT3-MMP has not been detected in breast tissue.139 Whereas MT4-MMP was identified in breast cancer cells, the role of this and MT5-MMP in breast cancer is not yet established,142 and MT6-MMP appears to be expressed predominantly by leukocytes.143

Increased expression of MMP-1 has been associated with lymph node metastases144 and poor prognosis145 in breast cancer, and was also one of the genes in the 70-gene expression signature identified by Van't Veer et al146 to predict distant metastases in lymph node-negative patients. MMP-1 is also implicated in mediating lung metastases in a mouse model of breast cancer.147 And, interestingly, elevated mRNA levels have been identified as a marker for predicting the development of invasive carcinoma from atypical ductal hyperplasia.148

Stromelysin 3 (MMP-11) was initially cloned as a gene differentially expressed in malignant compared to benign breast tissue149 and has been shown to be expressed exclusively by peri-tumoral fibroblasts.150-152 Expression levels of MMP-11, either by in-situ hybridization (ISH) or by immunohistochemistry (IHC), are associated with the presence of lymph node metastases,153 and both recurrence-free and overall survival;153-156 and in node-positive patients, elevated MMP-11 provides a strong independent prognostic parameter for disease-free survival.156

Collagenase 3 (MMP-13) was also first identified in breast carcinomas and has been localized predominantly to stromal cells of invasive carcinomas.157,158 In a recent study, including a series of ductal carcinoma in situ (DCIS) cases, MMP-13 was identified in the peri-ductal stroma of 7 of 8 cases exhibiting microinvasion, but not in 9 cases without microinva-sion.159 Although further studies are required to confirm this, it has been proposed that MMP-13 may play a pivotal role in the transition of DCIS to invasive disease, and may serve as a useful prognostic marker.

Finally, over recent years the impact of functional single nucleotide polymorphisms (SNPs) on modifying disease behavior has become evident. SNPs in the promoter region of several MMP genes influence levels of gene expres-sion.160-163 A study in our laboratory has shown that the 2G/2G genotype of MMP-1, which generates an increased level of gene expression, was more frequent in the lymph node-positive patients and conferred a 3.9-fold increased risk of lymph node metastasis, whilst the C/T genotype of MMP-9 was found to confer a 3.6-fold increased occurrence of lymph node metastasis.164 Przybylowska et al165 reported a similar association between the MMP-1 2G/2G

genotype and lymph node metastasis - in patients with breast cancer they showed no such association with the MMP-9 T allele. Such studies underline the biological role of MMPs in breast cancer and these genetic variations may help explain some of the individual variation observed in breast cancer behavior.

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