Immunohistochemical Visualisation of Cathepsin-D Expression in Breast Cancer

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1 Immunohistochemical Visualisation of Cathepsin-D Expression in Breast Cancer ELISABETH BARTHELL, IOANNIS MYLONAS, NAIM SHABANI, SUSANNE KUNZE, CHRISTINA KUHN, UDO JESCHKE and KLAUS FRIESE First Department of Obstetrics and Gynecology, Ludwig Maximilians University Munich, Maistrasse 11, München, Germany Abstract. Background: Cathepsin D (Cath-D), a lysosomal protease, is considered to be involved in the breakdown of the extracellular matrix during the process of tumour metastasis. Its expression in breast cancer in association with known factors of prognosis was investigated in this study. Materials and Methods: 120 breast cancer tumours were analysed immunohistochemically and evaluated with the immunoreactive score (IRS). Statistical evaluation was performed using the Mann- Whitney test (p<0.05). Results: For nodal-positive tumors we found a statistically significant increase of Cath-D immunohistochemical reaction. Ductal carcinomas showed a significantly higher immunohistochemical reaction compared to lobular carcinomas. We found a non-significant increase from G1 to G2 and G1 to G3, and a non-significant increase of Cath-D expression of all receptor-positive over all receptor-negative tumours. Conclusion: Cath-D expression was found to be associated with known prognostic factors. The clinical relevance of the observed difference depending on the histological type needs further investigation. Cath-D might be a useful marker to discriminate between ductal and lobular subtypes of breast cancer. Modulations of cell-to-cell and cell-to-matrix adhesion play important roles in the ability of breast cancer tumours to metastasize (1). In describing these adhesion-related antigens and proteolytic factors, one tries to find factors other than the known risk factors (grading, nodal status and receptor status) and to identify those patients who are at special risk for distant metastasis. These patients might especially benefit from systemic therapy. Cathepsin D (Cath-D), a lysosomal aspartyl-protease secreted by normal and malignant cells, is considered to be Correspondence to: Elisabeth Barthell, Ludwig-Maximilian- Universität München, Frauenklinik Innenstadt, Maistrasse 11, Muenchen, Germany. Tel: , Fax: , Elisabeth.barthell@med.uni-muenchen.de Key Words: Cathepsin D, breast cancer, immunohistochemistry. involved in the breakdown of the extracellular matrix. It was also demonstrated that it is crucial for fibroblast invasive outgrowth and could act as paracrine mediator between cancer and stromal cells (2). The extracellular secretion of Cath-D in nude mice is increased by estradiol and decreased by tamoxifen (3). High Cath-D expression has been shown to be an independent prognostic marker for metastasis of breast cancer when measured in tumour cytosol by several authors (4, 5), although some could not confirm this (6). A study from another group also confirmed the prognostic value of Cathepsin L (7). However, there are also conflicting results regarding the value of immunohistochemical evaluation of Cath-D in the tumour or in the stroma (7-10). The aim of this study was to determine the association of the degree of immunohistochemical staining of Cath-D with grading, nodal status, hormone receptors and histological type. Materials and Methods One hundred and twenty-one pure breast cancer tissue specimens (without carcinoma in situ) were obtained from the tumour bank of the First Department of Obstetrics and Gynecology of the LMU Munich. The patients had been treated between 1991 and The mean age was 62.3 years (±9.7; years), mainly postmenopausal. We investigated a total of 82 ductal and 39 lobular carcinomas. The grading of lobular carcinomas was determined ex post by a gynecopathologist, with the exception of 4 specimens due to technical reasons. There were 19.8% G1, 46.3% G2 and 30.6% G3 tumours (10.98%, 48.78% and 40.24% for ductal carcinomas and 38.46%, and 10.26% for lobular carcinomas, respectively). There were no significant differences with respect to nodal status and hormone receptors. Formalin-fixed paraffinembedded tissue slides (2-3 Ìm) were obtained from blocks. The sections were then incubated in methanol/h 2 O 2 (30 min) to inhibit endogenous peroxidase activity, washed in PBS (5 min) and treated with goat serum (20 min, 22ÆC) to reduce non-specific background staining. Incubation with the primary antibody (Table I) was carried out overnight at 4ÆC. Thereafter the slides were incubated with the biotinylated secondary anti-mouse antibody (1 h, 22ÆC) and avidin-biotinylated peroxidase (45 min, room temperature). Between each incubation, the sections were washed with /2007 $

2 Figure 1. Cathepsin-D IRS according to nodal status. Significantly higher expression was found in nodal positive tumours (p=0.048). Figure 2. Cathepsin-D IRS according to histology. Significantly higher Cath-D expression was found in ductal carcinomas (p=0.007). Table I. Antibody used in the study. Antibody Isotype Concentration Source C5 Mouse IgG 1 Ìg/ml Dianova phosphate-buffered saline (PBS, ph 7.4). Peroxidase staining reaction was carried out with diaminobenzidine/h 2 O 2 (1 mg/ml; 5 min) and stopped in tap water (10 min). Sections were counterstained in haematoxylin (1 min) and then cover-slipped. For positive controls, we used endometrial tissue of the secretory phase (see also (11)). There were no negative controls. 2036

3 Barthell et al: Cathepsin-D in Breast Cancer Figure 3. Cathepsin-D IRS according to grading. No significant differences in Cath-D expression. Figure 4. Cathepsin-D IRS according to hormone receptor status. Slight insignificant increase in Cath-D expression from receptor-negative to -positive tumours. The staining was evaluated using an immunoreactive score (IRS) described by Remmele and Stegner 1987 (12). It is calculated by optical staining intensity (+, ++ or +++) and the percentage of positively-stained cells (1-4). This was assesed in the tumour cells themselves, not in stromal tissue, by a gynecologist and revised by a biochemist experienced in immunohistochemistry evaluation. The SPSS/PC software package, version 11.0 (SPSS GmbH, München, Germany and IL, USA) was used for statistical analysis of all data. Results were evaluated using the nonparametric Mann-Whitney U-test for comparison and assessment of significant differences of the means. P 0.5 was considered to be significant. 2037

4 Figure 5. Lobular breast cancer GX N0 ER/PR-positive; x25; Immunohistochemistry with Cath-D Ab; IRS 4. Figure 6. Ductal breast cancer G2 N+ ER/PR-negative; x10; Immunohistochemistry with Cath-D Ab; IRS 9. Results For nodal-positive tumours we found a statistically significant increase of Cath-D IRS (p=0.048) (Figure 1). In histological subgroups this could only be demonstrated for the distribution of staining. There was also a significant difference in expression depending on the histological type. Ductal carcinomas exhibited significantly higher IRS than lobular ones (p=0.007) (Figure 2). With respect to grading, a non-significant increase from G1 to G2 and G1 to G3 for ductal carcinomas was found (Figure 3). Likewise an insignificant increase in Cath-D expression 2038

5 Barthell et al: Cathepsin-D in Breast Cancer from all receptor-positive over all receptor-negative tumours and in ductal carcinomas was found. In lobular carcinomas there was a minimal insignificant decrease (Figure 4). Discussion The immunohistochemical staining of Cath-D only resulted in a significant association with some known prognostic values (especially nodal status). This does not contradict those authors who describe Cath-D as an independent prognostic factor because a new prognostic or predictive factor is of particular value if it does not give the same information as known ones. Harbeck et al. (13) measured proteolytic factors in cytosol fractions from pulverised tumours and found only Cathepsin B (not Cath-D) to significantly correlate with nodal status. Moreover, they also found no strong correlation between proteolytic and established factors. In 1992, Maudelonde showed comparable results in quantifying Cath-D expression either by immunostaining or by cytosolic immuno-enzymatic assay (8). Still there are conflicting results between these two methods. In this study focused on immunohistochemistry, we found a difference depending on the histological type. Ductal carcinomas expressed higher amounts of Cath-D compared to lobular carcinomas. We found that almost all of the cases investigated showed positive Cath-D staining. We could not find a higher incidence of Cath-D staining in lobular carcinomas as described by Domagala et al. (14), but in fact found a higher staining intensity for this antigen in ductal carcinomas. This is in agreement with the work of Joensuu et al. (15) who found that Cath-D was more often present in the ductal than in the lobular histological type (80% vs. 54%, p=0.002), and its expression was strongly associated particularly with a high cell proliferation rate. Stromal Cath-D expression may reflect tumour differentiation and the functional status of ER in breast cancer, but stromal Cath-D and tumour metallothionine expression were the only factors found that may be of limited prognostic value (16). Therefore the clinical relevance of the observation described in our study needs further investigation. References 1 Loeffler G, Petrides P and Heinrich PC: Biochemie und Pathobiochemie. 7th ed. Heidelberg, Springer, pp , Liaudet-Coopman E, Beaujouin M, Derocq D, Garcia M, Glondu-Lassis M, Laurent-Matha V, Prebois C, Rochefort H and Vignon F: Cathepsin D: newly discovered functions of a longstanding aspartic protease in cancer and apoptosis. Cancer Lett 2: , Dabrosin C, Johansson AC and Ollinger K: Decreased secretion of Cathepsin D in breast cancer in vivo by tamoxifen: mediated by the mannose-6-phosphate/igf-ii receptor? 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