Intraepithelial matrix metalloproteinase-9 in invasive breast carcinoma, normal tissue adjacent to tumor, and normal mammoplasty specimens: a comparative study considering relationship between matrix metalloproteinase-9 and some clinicopathologic prognostic factors of breast carcinoma CURRENT

Background- Matrix metalloproteinase-9 (MMP-9), an enzyme with major role in remodeling of extracellular matrix, has been the focus of attention in some previous studies in the field of breast cancer. We conducted a comparative study to investigate the frequency of this marker in tissue specimens of invasive breast carcinoma, tumor control, and normal control. The relationship between matrix metalloproteinase-9 and some clinicopathologic factors of breast carcinoma has also been evaluated. Methods- Formalin-fixed and paraffin-embedded tissue specimens from three groups including 40 invasive breast carcinoma (tumor group) and their adjacent normal tissue (tumor control), as well as 40 normal mammoplasty specimens (normal control) were studied. The samples were from the pathology archive of Alzahra Hospital, Isfahan, Iran, from 2016 to 2018. The status of intraepithelial MMP-9 was studied and compared in these three groups using immunohistochemistry. The relationship between intraepithelial MMP-9 and some clinicopathologic prognostic factors was also evaluated in tumor group. Results - The extent of intraepithelial MMP-9 immunostaining in all positive specimens was 100%. The results of intraepithelial MMP-9 staining intensity was as follow: 12.5% strong, 27.5% moderate, 27.5% mild, and 32.5% negative in tumor group; 17.5% strong, 22.5% moderate, 32.5% mild, and 27.5% negative in tumor control group; and 10% strong, 40% moderate, 27.5% mild, and 22.5% negative in normal control group. Intraepithelial MMP-9 immunostaining intensity showed significant difference between tumor and tumor control groups (P<0.001). Intraepithelial MMP-9 immunostaining intensity showed no significant difference between tumor and normal control groups, and between tumor control and normal control groups (P>0.05).

Cancer registry reports indicate breast cancer as the most prevalent malignant tumor among Iranian women (4). There are definitive risk factors for breast cancer including early menarche, late menopause, nulliparity, and positive familial history. However, several molecules have been the focus of attention as probable determinant factors of breast cancer behavior and outcome in recent years (5,6).
Matrix metalloproteinases (MMPs) are a huge family of extracellular enzymes dependent to zinc and calcium. These enzymes are categorized based on their function and structures into matrilysin, gelatinases, stromelysin, and collagenases (7).
MMP-9 is one of the most important gelatinases which is highly effective in tumoral invasion and destruction of type IV collagen and other components of extracellular matrix (8,9).
There is low expression of MMPs in normal tissues. Increased activity and expression of these enzymes causes tissue destruction. This situation occurs in inflammatory diseases and tumors. As a result, MMPs have been considered as a marker for tissue destruction (10).
Early diagnosis of breast cancer and its micro-metastases are pivotal factors in cancer treatment.
Some studies have found relationship between MMP-9 expression and prevalence of metastasis in breast carcinoma (11,12). Stromal MMP-9 expression has been shown as a predicting factor for hormonal response and survival in patients with breast cancer (13). The relationship between MMP-9 expression and lymph node status has also been reported in patients with breast cancer (14).
However, the results of previous studies on the issue of MMP-9 expression in breast cancer and its impact on tumor prognosis has not been yet conclusive. The objective of this study has been to assess the frequency of intracellular expression of MMP-9 in invasive breast carcinoma and normal breast tissue, and to investigate the relationship between intracellular expression of this marker in breast carcinoma and some well-known clinicopathological prognostic variables of this cancer.

Methods:
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.
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 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.

Results:
In this study, we evaluated 40 specimens of invasive breast carcinoma (tumor group) and normal tissue adjacent to these carcinoma specimens (tumor control group), as well as 40 normal control specimens from mammoplastic surgery (normal control group). The extent of intraepithelial MMP-9 immunostaining in all positive specimens was 100%. The results of intraepithelial MMP-9 staining intensity was as follow: 12.5% strong, 27.5% moderate, 27.5% mild, and 32.5% negative in tumor group; 17.5% strong, 22.5% moderate, 32.5% mild, and 27.5% negative in tumor control group; and 10% strong, 40% moderate, 27.5% mild, and 22.5% negative in normal control group (Table 1) ( Fig. 1). Intraepithelial MMP-9 immunostaining intensity showed significant difference between tumor and tumor control groups (P < 0.001) ( Table 2). Intraepithelial MMP-9 immunostaining intensity showed no significant difference between tumor and normal control groups, and between tumor control and normal control groups (P > 0.05).   is highly increased in patients with breast cancer compared to normal population. This increase was also associated with lymph node metastasis, higher tumor grade, shorter relapse-free period, and reduced overall survival (29). Li et al found MMP-9 and MMP-2 markers to have high prognostic value in lymph node negative breast carcinoma (30). Mylona et al suggested the possible relationship between cytoplasmic MMP-9 and well-differentiated breast carcinoma, while they found stromal MMP-