The immunohistochemical expression of desmoplakin and its role in vivo in the progression and metastasis of breast cancer

Abstract

Desmoplakin (DP) is a protein located at the inner plaque of desmosomes where it associates with the desmosomal cadherins to form a cell adhesion complex. Reduced expression of DP has been correlated with the progression of several cancers, but its role in in vivo breast cancer is yet to be established. The aim of this present paper was to determine the immunohistochemical (IHC) expression of DP in breast cancer specimens (n=75) in comparison with ductal carcinoma in situ (DCIS) (n=26), tumour associated normal (n=29) and normal breast tissue (n=7). DP expression was correlated with that of desmosomal cadherin, Desmoglein 2 (Dsg2) and other clinical and IHC prognostic markers. DP staining occurred at the sub-plasma membrane level. There was no significant correlation between the level of DP (as assessed by the H-score) and that of Dsg. Significantly stronger staining was demonstrated in normal breast tissue and well differentiated tumours compared with more moderately or poorly differentiated tumours (P=0.04). A significant inverse correlation was seen between DP staining and tumour size (P=0.01). In a limited series of 8 cases, primary tumours demonstrated significantly stronger staining than the matched metastatic lymph nodes (P=0.046). Of all the IHC markers examined, only Ki-67 showed a significant inverse relationship with DP staining (P=0.01). In summary, the data suggest that loss of DP may be of potential importance in progression of breast cancer in vivo from normal, DCIS, well differentiated through to poorly differentiated, large tumours. In addition, this loss may be associated with metastasis.

Introduction

Breast cancer affects approximately 1 in 12 women in the U.K. The most devastating aspect of cancer is the spread from the primary site to distant organs. Despite the ever increasing advances in ‘modern’ treatment modalities, such as surgical, chemo- and radiotherapy, the presence of metastasis is the major cause of treatment failure for cancer patients1, 2. Approximately 30% of patients with newly diagnosed tumours already have clinically detectable metastases at the time of presentation. Furthermore, of those 70% of patients free of clinically detectable metastases, only 50% can be cured with local irradiation. The remainder have clinically occult metastases which manifest at a later date[2]. Local invasion and subsequent distant metastasis is a complex multistep process3, 4, 5. The initial step in this ‘metastatic cascade’ is the loss of cell–cell adhesion at the primary site. Reduced expression of several cell adhesion molecules, including E-cadherin6, 7, 8, 9, 10and their associated desmosomal cadherins including Desmoglein 2 (Dsg2)11, 12, 13, 14have been implicated in this process. We recently reported a loss of Dsg2 expression in vivo in breast cancer in comparison with both normal breast tissue and ductal carcinoma in situ and an association in vitro with decreased cell–cell aggregation and increased invasion and motility[15].

Desmosomes play a critical role in the development and maintenance of epithelial tissue integrity. They are composed of a pair of disc-like intercellular junctions, usually less than 0.5 μm in diameter. Their functions include cell–cell adhesion and maintenance of cell integrity via their attachments to the intermediate filaments[16]. Desmosomes occur in all epithelia, cardiac muscle, the arachnoid and piamater and the follicular dendritic cells of the lymphoid system17, 18, 19, 20.

The plaque region of the desmosome is thought to link the intermediate filaments (IFs) to the cell surface. Plaque-associated proteins are located at the inner plaque region and possess the ability to interact both with the IFs and the components of the plaque. The two major plaque-associated proteins are desmoplakin (DP) and plakoglobin (PG). DP I and II are a pair of proteins (molecular weights 250 000 and 215 000 Da, respectively), derived from the same DP gene. DP1 is present in all desmosome-bearing tissues[21], whilst DP2 is more variably expressed, found in lower levels of non-stratified tissues and absent in certain tissue, such as the heart[22].

Several electron microscopical studies of desmosomes have suggested that a reduction in desmosomes is associated with invasive behaviour23, 24. These initial studies were followed by immunohistochemical examination of the expression of the different desmosomal components in vivo, demonstrating a marked reduction in the expression of both desmoplakin and desmoglein in several carcinomas, including transitional cell carcinomas of the urinary tract[11], squamous cell carcinomas of the oral cavity[13]and adenocarcinoma of the uterine cavity[14]. These studies correlated expression with degree of differentiation and lymph node involvement, demonstrating a DP loss associated with local progression and metastasis. Several studies both in vitro[25]and in vivo[26]have demonstrated that loss of plakoglobin expression in breast cancer is related to invasive behaviour. In breast cancer, data on the role of DP are limited and inconclusive. To date, only one study has examined DP expression in breast cancer in vivo and has surprisingly failed to demonstrate any association with disease progression, although no lymph node data were available to determine its role in metastasis[27]. It is, therefore, apparent that further research is required to establish the role of DP in breast cancer.

Several clinical and immunohistochemical parameters are used as clinical guidelines for assessing the aggressiveness of tumours in terms of potential metastatic ability and cause of death. In the literature, size, grade and lymph node involvement on multivariate analysis are prognostic indicators28, 29. Similarly immunohistochemical markers are good indicators of survival and response to certain adjuvant treatments. Patients with oestrogen receptor (ER) positive tumours have prolonged disease-free survival after primary treatment and superior overall survival compared with ER negative patients[29]. Veronese and associates[30]reported that Ki-67 levels predicted 4-year survival independently of ER and lymph node status. High Ki-67 levels have been linked with tumour aggressiveness and disease spread[31]. Finally epithelial growth factor receptor (EGFR) expression has been found to be significantly associated with presence and number of lymph node metastasis[32]and long-term outcome[33].

The aim of this present study was thus to assess the role of desmoplakin I/II in breast cancer progression by comparing the immunohistochemical staining in breast cancer with ductal carcinomas in situ (DCIS) and normal breast tissue, and to assess DP’s potential role in invasion and metastasis by correlating its expression with a range of clinical (size, grade and lymph node status) and relevant immunohistochemical markers (Dsg 2, Ki-67, EGFR and ER), as well as examining a small available group of matched primary breast cancers with their lymph node metastasis.