ANTICANCER RESEARCH 27: 2035-2040 (2007) 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, 80337 München, Germany Abstract. Background: Cathepsin D (Cath-D), a lysosomal involved in the breakdown of the extracellular matrix. It was protease, is considered to be involved in the breakdown of the also demonstrated that it is crucial for fibroblast invasive extracellular matrix during the process of tumour metastasis. Its outgrowth and could act as paracrine mediator between expression in breast cancer in association with known factors cancer and stromal cells (2). The extracellular secretion of of prognosis was investigated in this study. Materials and Cath-D in nude mice is increased by estradiol and decreased Methods: 120 breast cancer tumours were analysed by tamoxifen (3). High Cath-D expression has been shown immunohistochemically and evaluated with the immunoreactive to be an independent prognostic marker for metastasis of score (IRS). Statistical evaluation was performed using the Mann- breast cancer when measured in tumour cytosol by several Whitney test (p<0.05). Results: For nodal-positive tumors we authors (4, 5), although some could not confirm this (6). A found a statistically significant increase of Cath-D study from another group also confirmed the prognostic immunohistochemical reaction. Ductal carcinomas showed a value of Cathepsin L (7). However, there are also conflicting significantly higher immunohistochemical reaction compared to results regarding the value of immunohistochemical lobular carcinomas. We found a non-significant increase from G1 evaluation of Cath-D in the tumour or in the stroma (7-10). to G2 and G1 to G3, and a non-significant increase of Cath-D The aim of this study was to determine the association expression of all receptor-positive over all receptor-negative of the degree of immunohistochemical staining of Cath-D tumours. Conclusion: Cath-D expression was found to be with grading, nodal status, hormone receptors and associated with known prognostic factors. The clinical relevance histological type. of the observed difference depending on the histological type needs further investigation. Cath-D might be a useful marker to Materials and Methods discriminate between ductal and lobular subtypes of breast cancer. One hundred and twenty-one pure breast cancer tissue specimens (without carcinoma in situ) were obtained from the tumour bank Modulations of cell-to-cell and cell-to-matrix adhesion play of the First Department of Obstetrics and Gynecology of the LMU important roles in the ability of breast cancer tumours to Munich. The patients had been treated between 1991 and 2000. metastasize (1). In describing these adhesion-related antigens The mean age was 62.3 years (±9.7; 46-84 years), mainly and proteolytic factors, one tries to find factors other than postmenopausal. We investigated a total of 82 ductal and 39 the known risk factors (grading, nodal status and receptor lobular carcinomas. The grading of lobular carcinomas was status) and to identify those patients who are at special risk determined ex post by a gynecopathologist, with the exception of 4 for distant metastasis. These patients might especially benefit 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 from systemic therapy. carcinomas and 38.46%, 41.03 and 10.26% for lobular carcinomas, Cathepsin D (Cath-D), a lysosomal aspartyl-protease respectively). There were no significant differences with respect to secreted by normal and malignant cells, is considered to be nodal status and hormone receptors. Formalin-fixed paraffin- embedded tissue slides (2-3 Ìm) were obtained from blocks. The sections were then incubated in methanol/H2O2 (30 min) to inhibit endogenous peroxidase activity, washed in PBS (5 min) and treated Correspondence to: Elisabeth Barthell, Ludwig-Maximilian- with goat serum (20 min, 22ÆC) to reduce non-specific background Universität München, Frauenklinik Innenstadt, Maistrasse 11, staining. Incubation with the primary antibody (Table I) was 80337 Muenchen, Germany. Tel: +49 8951904695, Fax: +49 89 carried out overnight at 4ÆC. Thereafter the slides were incubated 51604696, e-mail: [email protected] with the biotinylated secondary anti-mouse antibody (1 h, 22ÆC) and avidin-biotinylated peroxidase (45 min, room temperature). Key Words: Cathepsin D, breast cancer, immunohistochemistry. Between each incubation, the sections were washed with 0250-7005/2007 $2.00+.40 2035 ANTICANCER RESEARCH 27: 2035-2040 (2007) 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. phosphate-buffered saline (PBS, pH 7.4). Peroxidase staining reaction was carried out with diaminobenzidine/H2O2 (1 mg/ml; 5 Antibody Isotype Concentration Source min) and stopped in tap water (10 min). Sections were counterstained in haematoxylin (1 min) and then cover-slipped. For C5 Mouse IgG 1 Ìg/ml Dianova positive controls, we used endometrial tissue of the secretory phase (see also (11)). There were no negative controls. 2036 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) The SPSS/PC software package, version 11.0 (SPSS GmbH, described by Remmele and Stegner 1987 (12). It is calculated by München, Germany and IL, USA) was used for statistical optical staining intensity (+, ++ or +++) and the percentage of analysis of all data. Results were evaluated using the non- positively-stained cells (1-4). This was assesed in the tumour cells parametric Mann-Whitney U-test for comparison and assessment themselves, not in stromal tissue, by a gynecologist and revised by a of significant differences of the means. P≤0.5 was considered to biochemist experienced in immunohistochemistry evaluation. be significant. 2037 ANTICANCER RESEARCH 27: 2035-2040 (2007) 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 difference in expression depending on the histological type. Ductal carcinomas exhibited significantly higher IRS than For nodal-positive tumours we found a statistically lobular ones (p=0.007) (Figure 2). significant increase of Cath-D IRS (p=0.048) (Figure 1). In With respect to grading, a non-significant increase from G1 histological subgroups this could only be demonstrated for to G2 and G1 to G3 for ductal carcinomas was found (Figure the distribution of staining. There was also a significant 3). Likewise an insignificant increase in Cath-D expression 2038 Barthell et al: Cathepsin-D in Breast Cancer from all receptor-positive over all receptor-negative tumours 4 Spyratos F, Maudelonde T, Brouillet JP, Brunet M, Defrenne A, and in ductal carcinomas was found. In lobular carcinomas Andrieu C, Hacene K, Desplaces A, Rouesse J and Rochefort H: there was a minimal – insignificant – decrease (Figure 4). Cathepsin D: an independent prognostic factor for metastasis of breast cancer. Lancet 8672: 1115-1118, 1989. 5 Harbeck N, Harbeck N, Alt U, Berger U, Krueger A, Thomssen Discussion C, Jaenicke F, Hoefler H, Kates RE and Schmitt M: Prognostic impact of proteolytic factors (urokinase-type plasminogen The immunohistochemical staining of Cath-D only resulted activator, plasminogen activator inhibitor 1, and cathepsins b, d, in a significant association with some known prognostic and l) in primary breast cancer reflects effects of adjuvant values (especially nodal status). This does not contradict systemic therapy. Clin Cancer Res pp. 2757-2764, 2001. 6 Korkolis DP, Tsoli E, Fouskakis D, Yiotis J, Koullias GJ, those authors who describe Cath-D as an independent Giannopoulos D, Papalambros E, Nikiteas NI, Spiliopoulou CA, prognostic factor because a new prognostic or predictive Patsouris E, Asimacopoulos P and Gorgoulis VG: Tumor factor is of particular value if it does not give the same histology and stage but not p53, Her2-neu or cathepsin-D information as known ones. expression are independent prognostic factors in breast cancer Harbeck et al. (13) measured proteolytic factors in cytosol patients. Anticancer Res 24(3B): 2061-2068, 2004. fractions from pulverised tumours and found only Cathepsin 7 Kandalaft PL, Chang KL, Ahn CW, Traweek ST, Mehta P and B (not Cath-D) to significantly correlate with nodal status. Battifora H: Prognostic significance of immunohistochemical analysis of cathepsin D in low-stage breast cancer. Cancer 9: 2756- Moreover, they also found no strong correlation between 2763, 1993. proteolytic and established factors. In 1992, Maudelonde 8 Maudelonde T, Brouillet JP, Roger P, Giraudier V, Pages A and showed comparable results in quantifying Cath-D Rochefort H: Immunostaining of cathepsin D in breast