Elsevier

European Journal of Cancer

Volume 38, Issue 18, December 2002, Pages 2362-2370
European Journal of Cancer

Immunohistochemical expression of extracellular matrix components tenascin, fibronectin, collagen type IV and laminin in breast cancer: their prognostic value and role in tumour invasion and progression

https://doi.org/10.1016/S0959-8049(02)00210-1Get rights and content

Abstract

The immunohistochemical expression of the extracellular matrix (ECM) components tenascin (TN), fibronectin (FN), collagen type IV (Coll) and laminin (LN), and their possible relationships were studied in a series of 134 operable breast cancer cases. Their expression was also compared with the expression of the proteolytic enzyme cathepsin D (CD), the adhesion molecule CD44 standard form (CD44s) and other known factors to clarify the prognostic value and role of these molecules in tumour progression and metastasis. TN expression in the tumour stroma was positively correlated with tumour grade and size, CD44s expression, tumour and stromal CD expression as well as with FN, laminin and Coll expression in the same areas. TN expression was inverse correlated with ER status. Its expression at the invasion front was only positively correlated with the lymph node status. Survival analysis showed an increased mortality risk associated with high levels of TN expression. In multivariate analysis, among the ECM proteins, only TN expression was independently correlated with patients' survival. FN expression was positively correlated with lymph node involvement, with the proliferation-associated index Ki-67 and stromal CD expression. Survival analysis showed an increased mortality risk associated with a high level of FN expression. Coll expression was positively correlated with the tumour size and LN expression. An inverse relationship of Coll expression with ER and PgR receptor status was also found. LN expression was positively correlated with tumour and stromal CD expression, with the proliferation-associated index Ki-67 and inversely with ER receptor status. The observed alterations in the expression of ECM proteins in breast cancer tissue and their correlations with the proteolytic enzyme CD and the adhesion molecule CD44s, suggest an involvement in cancer progression. In addition, overexpression of stromal TN and FN seems to have negative prognostic value in breast cancer patients.

Introduction

Cancer cells are surrounded by a modified extracellular matrix (ECM) composed of a complex meshwork of collagens, fibrillar glycoproteins and proteoglycans which intercommunicate with the cell interior and thus modulate cell adhesion, proliferation and differentiation [1]. Tenascin (TN) and fibronectin (FN) are glycoprotein components of the ECM which seem to have competitive functions. It can be speculated that this competitive relationship between these molecules is important for cellular functions [2]. TN is a protein of the ECM that contains 14 repeats of the epidermal growth factor (EGF)-like domain. It is produced by stromal fibroblasts and also by epithelial cells of normal and malignant breast tissues and is expressed transiently during embryogenesis, inflammation and malignancy [3]. Recently, a new class of insoluble low affinity matrix-tethered ligand for the epidermal growth factor-receptor (EGFR) was recognised within the EGF-like repeats of tenascin-C [4].

In adult breast tissue, TN expression is menstrual cycle dependent [5] and it has a different pattern of distribution in normal, benign and malignant breast tissue. This is variably present in the basement membrane region of breast epithelia and is enhanced periductally next to the basement membrane in intraductal carcinoma of the breast 6, 7, 8. In infiltrating breast carcinoma, TN is expressed in the stroma and appears to be associated with progressive disease and poor prognosis 9, 10, 11, 12, 13.

FN is regarded as the major mesenchymal ECM glycoprotein involved in cell–matrix and cell–cell adhesion, cell migration, morphogenesis, differentiation and oncogene transformation [14]. Tissue FN is found in the connective tissue in close apposition to the BMs. Studies of FN in breast carcinomas showed a strong expression and different distribution compared with normal breast parenchyma 15, 16.

Laminin and collagen type IV constitute the major intrinsic components of BMs and are involved in cellular adhesion to BMs and the extracellular matrix. Proteolytic enzymes, form a cascade system to facilitate the breakdown of the extracellular matrix, including the BM as the initial event 17, 18. It has been showed that type IV collagen is involved in regulation of mammary cell proliferation, cell attachment and migration [19]. Laminin has been reported to play a role in the adhesion of the cell to the matrix. Invading tumour cells are capable of attachment to the matrix through specific laminin receptors present on epithelial cell membranes and of protease release in the interstitial, promoting BM disruption and cell diffusion [20]. Laminin, like FN, has been shown to play an important role in regulating cell migration, facilitating tumour invasion [21]. Immunohistochemical studies of the distribution of laminin in human breast carcinomas and its role in the process of tumour invasion, have been reported 21, 22. However, cathepsin D (CD) is an ostrogen-induced lysosomal enzyme that can act either directly, by digesting the extracellular matrix, or indirectly by initiating the proteolytic cascade, that may be responsible for the breakdown of BM components [23]. In addition, current evidence suggest that CD44 proteins participate in a large number of related molecular processes, which involve specific adhesions to hyaluronate, collagen and fibronectin [24] and cell migration [25].

In this study, we assessed the interrelationship of the expression of extracellular matrix components TN, FN, collagen type IV and laminin in a series of invading breast cancer in order to elucidate the role of these molecules in tumour expansion. Their expression was also correlated with the expression of proteolytic enzyme Cathepsin D (CD) and with the adhesion molecule CD44s, in order to obtain a better understanding of the role of these molecules in breast tumour invasion and progression. In addition, their expression were correlated with other known potential prognostic factors (ER, PgR, Ki-67 and proliferating cell nuclear antigen (PCNA) and clinicopathological parameters (tumour grade and size, lymph node status, distant metastasis, overall survival) in an attempt to clarify their prognostic importance for clinical implications. In 24 cases, the immunohistochemical expression of extracellular matrix components was also studied in the lymph node involved tissue.

Section snippets

Materials and methods

A cohort of 138 patients with primary invasive breast carcinoma who were surgically resected were investigated. For 82 of patients, we had follow-up data and these patients were included in the survival analysis. All patients had a mastectomy with axillary lymph node dissection performed as indicated and were followed-up regularly at the Medical Oncology Department of University Hospital of Ioannina. The patients' ages ranged from 28 to 91 years (mean±SD age 55.5±13.8 years). The median

Tenascin immunostaining

In the normal breast, a thin, focally discontinuous band of TN immunoreactivity was usually noted around ducts and acini; no appreciable reaction was observed beyond the immediate periductal and periacinar stromal regions, except for occasional thin bands around vessels. Intralobular and interlobular stroma was usually negative in fibroadenomas and fibrocystic disease.

In carcinomas, there was an increased TN immunoreactivity in comparison to normal gland that surrounded the cancer nests and in

Discussion

For a long time, only neoplastic cells were the focus of interest in cancer research, while the stroma was thought to lack any major biological and clinical significance. However, it has now become clear that stromal cells and their products play a significant role in the phenotype of cancer cells 6, 7, 10, 14, 20. Thus, tumours present a complex ecosystem in which interactions between tumour cells and the extracellular matrix, as well as host cells, lead to reciprocal influences resulting in

Acknowledgements

We offer our thanks to Mrs. A. Christodoulou for her technical assistance.

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