Expression of Metalloproteinases MMP-2 and MMP-9 in Sentinel Lymph Node and Serum of Patients with Metastatic and Non-Metastatic Breast Cancer

Materials and Methods

Fifty patients with metastatic and non-metastatic breast cancer undergoing mastectomy or quadrantectomy surgery at ‘Giovanni Paolo II’ National Cancer Institute of Bari, Italy, were enrolled between 2004 and 2007. A control group comprised 34 healthy women who were asked to consent to participate in the study.

All the data obtained from medical records, after informed consent of the patients enrolled was given, were included in a database developed in Microsoft Excel (Microsoft Corp, WA, USA). Cytological and histopathological parameters considered were the following: histological diagnosis of cancer, menopausal status, cancer family, TNM, G, ER and PgR status), MIB-1, expression of HER-2/Neu and type of therapy.

The expression of gelatinase A and B in paraffin-embedded sentinel lymph nodes was evaluated using immunohistochemistry, while the serum concentrations of MMP-2 and MMP-9 were tested using zymography.

Identification of sentinel lymph nodes by perilesional lymphoscintigraphy. Colloidal particles of human albumin, measuring between 20 and 80 nanometers (Nanocol) labeled with ^99mTc were used. The day before surgery for mastectomy or quadrantectomy, the solution containing the radioactive tracer (0.2 mCi 99 mTc in a volume of 0.2-0.4 saline) was inoculated with 25 G needle by injection under the skin at the breast lesion for 4 times. The patient was asked to massage the area of inoculation for several minutes in order to speed up the lymphatic drainage. Subsequently, dynamic lymphoscintigraphy was performed by gamma camera (Siemens Diacam. LabInstruments Inc., India). Scintigraphic images were acquired for about 10-20 minutes until the identification of the route of lymphatic drainage duct and its sentinel node. The anterior oblique projection at 45°C is optimal because it allows the injection site to be better distinguished from the axillary sentinel lymph node. The place of accumulation corresponding to LS was marked with a dermographic pen.

Expression of gelatinase A and B in lymph nodes by immunohistochemistry. All lymph nodes were dissected clearly and fixed in formalin. The sentinel lymph node was bisected along its major axis and embedded in paraffin. For each paraffin block obtained, 40 pairs of sections, each 4 μm-thick, were cut at 50 μm intervals. If residual tissue was left, an additional pair of sections was cut at 100 μm intervals until the lymph node had been sampled completely. One section of each pair was stained with haematoxylin and eosin. If the result of the staining was ambiguous, the parallel section was stained for cytokeratin.

Immunostaining for MMP-2 and MMP-9 was performed on the formalin-fixed tissue sections. Sections were deparaffinised and heated in a microwave oven for 10 min for unmasking using an antigen retrieval protocol (Benchmark XT; Ventana). (Medical System Inc., Tucson, Arizona, USA).

Sections were immersed in 3% hydrogen peroxide in 100% methanol for 10 min to block the endogenous peroxidase activity. All sections were incubated with primary antibody MMP-2 (clone 75-7F7 mouse monoclonal antibody; Calbiochem-Darmstadt Germany; 1:100) and MMP-9 (clone 56-2A4 monoclonal antibody; Calbiochem; 1:100) for 60 min at room temperature. Subsequently the sections were incubated with biotinylated anti-mouse (i-View DAB Detection Kit; Ventana) for 20 min and treated with peroxidase4-conjugated streptavidin for 20 min (i-View DAB Detection Kit).

The sections were then immersed into 3-amino-9-ethyl-carbazole (AEC, Tucson, AZ, USA) solution. The slides were counterstained with haematoxylin solution, dehydrated and mounted.

Between steps, the slides were washed three times with phosphate-buffered saline (PBS). PBS was used as a negative control instead of the primary antibody.

Hormone receptor expression (IHC). ER and PgR assays were performed with primary monoclonal antibodies to the receptors (anti-human oestrogen receptor α1D5; Dako Diagnostics, Denmark, and anti-human progesterone receptor 1A6; YLEM s.r.l, Rome, Italy). Cases of intraductal infiltrating carcinoma (IDC) with known reactivity for the antibody were used as positive controls. Sections 4-6 μm-thick obtained from the paraffin-embedded tissues were placed on slides, pretreated with endogeneous peroxidase and blocked with hydrogen peroxide. Then antigen retrieval was performed using a microwave at 650 W for 3 cycles of 8 minutes each and citrate buffer 1×, 0.07 M, pH 6±1. Subsequently, the slides were incubated, firstly with streptavidin-biotinylated ABC (StreptABComplex) overnight at 4°C, and then with amino-ethyl-carbazole (AEC) as chromogen. Slides were examined under light microscopy. The results were recorded as the percentage of positively stained target cells with positive samples being defined as those showing more than 10% stained tumour cell nuclei.

HER–2/Neu expression (immunohistochemistry). HER-2/Neu immunostaining was performed on 3-4 μm-thick sections obtained from the paraffin-embedded tissues, using the polyclonal antibodies A0485 (Hercept Test, Dako Diagnostics) directed against the C-terminal fragment of the molecule (intra-domain) and diaminobenzidine tetrahydrochloride (DAB) as chromogen. Avidin-biotin peroxidase complex was used as standard. Cases of IDC with known reactivity for the antibody were used as positive controls. Negative control reactions were performed omitting the antibody. Using light microscopy, breast carcinoma cells that showed intense membrane staining were considered to be positive, while those that showed cytoplasmic staining in the absence of membrane staining were skipped.

Membrane immunoreactivity and staining patterns were evaluated and scored using a scale of 0 to +3 according to the scoring system outlined in Dako's Hercept Test manual. The first step in the scoring system was to ascertain whether more than 10% of the tumour cells had complete membrane positivity and, finally, the staining intensity was graded with 1 if membrane staining was absent or very weak intensity, with 2, if the membrane staining intensity was weak, or with +3, if the membrane staining intensity was strong.

Gelatinolytic zymography assay. Gelatinolytic zymography was performed as previously described (16). Briefly, patients' sera were analysed for gelatin degradation activity by 8% SDS-PAGE containing 5.8 mg/ml gelatin under non-reducing conditions. Electrophoresis was performed and, after a brief wash with water, SDS was removed from the gel by incubation with 2% Triton X-100/PBS solution. Gelatinolytic activities were developed in a buffer containing 5 mM CaCl₂, 150 mM NaCl, and 50 mM Tris at 37°C for 16 h and then visualised by staining the gel with Coomassie Blue R-250. The amounts of MMP-2 and MMP-9 were quantified using scanning densitometry with an image analysis software (Image Master ID Prime; Pharmacia Biotech, UK) (16).

Statistical analyses. The data were analysed using the unpaired Student's t-test and the MannWhitney U-test. The frequency distribution of MMP-2 and MMP-9 in lymph node and serum were assessed using univariate ANOVA. Pearson's correlation analysis was performed between the two MMPs. All statistical analyses were performed by SPSS for Windows v. 9.01 (SPSS Inc., Chicago, IL, USA). Numerical data are expressed as mean±standard deviation (SD). P-values≤0.05 were considered to be statistically significant.