This cross-sectional study was performed on formalin-fixed and paraffin-embedded tissue blocks from 40 invasive breast carcinomas (tumor group) and normal tissue adjacent to carcinoma (tumor control group), as well as 40 normal breast tissue specimens from mammoplastic surgery (normal control group). The specimens were obtained from pathology archive of Alzahra Hospital, Isfahan, Iran, from 2016 to 2018. The study was approved by ethical committee of Isfahan University of Medical Sciences, Isfahan, Iran (Ethical code: IR.MUI.MED.REC.1398.055). Inclusion criteria were breast lumpectomy or mastectomy specimens with documented diagnosis of invasive breast carcinoma having normal tissue adjacent to carcinoma and dissected axillary lymph nodes. Concerning normal control group, only mammoplasty specimens having normal breast tissue without any kind of breast pathology were included in the study. Exclusion criteria were those carcinoma specimens without adjacent normal breast tissue and/or lacking dissected axillary lymph nodes. Concerning normal control group, mammoplasty specimens with any kind of breast pathology were excluded from the study.
We used immunohistochemistry to stain all 120 specimens with MMP-9 antibody (Rabbit Polyclonal Antibody, Diagnostic BioSystems, Canada). Five micron sections were prepared from tissue blocks and underwent immunohistochemical staining according to the following steps:
48 hours incubation in oven at 37 °C, dewaxation by 100% xylol, rehydration by decreasing concentrations of ethanol (100%, 85%, and 75%), rinsing in 10% phosphate-buffered saline (PBS) solution, 30 minutes incubation in 10% H2O2 and methanol to inhibit endogenous peroxidase activity, rinsing in 10% PBS solution, 14 minutes incubation in citrate-buffered solution (PH = 6.1) in microwave, rinsing in 10% PBS solution, adding blocking serum for 30 minutes to block endogenous non-specific bindings, drying, adding primary monoclonal antibody, 30 minutes incubation at room temperature, rinsing in 10% PBS solution, adding broad-spectrum secondary antibody for 30 minutes, adding horseradish peroxidase-streptavidin and diaminobenzidine (DAB) for 30 minutes and 10 minutes, respectively, rinsing in 10% PBS solution, dehydration by increasing concentrations of ethanol (75%, 85%, and 100%), and counterstaining with hematoxylin.
The intensity and extent of cytoplasmic MMP-9 immunoreactivity + were then examined in epithelial cells of breast carcinoma and normal breast tissue. Epithelial cells, fibroblasts, and extracellular matrix all may show immunoreactivity with MMP-9 antibody (13, 14). However, only MMP-9 expression in epithelial cells has been considered in this study. Scores of staining intensity were defined as follow (13–16):
Score 0: Negative
Score 1: Mild
Score 2: Moderate
Score 3: strong
The percentage of stained epithelial cells irrespective of the intensity of staining was evaluated to determine and classify staining extent as follow (13–16):
Score 0: 0 to 10%
Score 1: 11 to 25%
Score 2: 26 to 50%
Score 3: 51 to 75%
Score 4: 76 to 100%
The intensity and extent of cytoplasmic staining of MMP-9 in epithelial cells was then compared between the three groups. In tumor group, the relationship between intraepithelial MMP-9 expression and some prognostic factors including age, tumor size, tumor grade, and lymph node status was also studied. Data concerning age, tumor size (greatest tumor diameter), tumor grade, and lymph node status were all achieved from pathology archive of the hospital. SPSS software, version 24, was used for data analysis. Data were shown as frequency, mean, and standard deviation (SD). To determine the relationship between MMP-9 expression and prognostic variables in tumor group, Chi-Square and one-way ANOVA were used. P-value less than 0.05 was considered as significant.