|Year : 2022 | Volume
| Issue : 3 | Page : 280-286
Expression of matrix metalloproteinase-2 and survivin in pancreatic carcinoma: an immunohistochemical study
Said Abu-Alkhair Mohamed, Yasser Mohamed Eldowik
Department of Pathology, Al-Azhar university, Faculty of medicine, Assuit, Egypt
|Date of Submission||05-May-2021|
|Date of Decision||17-Feb-2022|
|Date of Acceptance||06-Mar-2022|
|Date of Web Publication||11-Oct-2022|
MD Said Abu-Alkhair Mohamed
Department of Pathology, Faculty of Medicine, Al-Azhar University, Assuit 44628
Source of Support: None, Conflict of Interest: None
Background and Aim Matrix metalloproteinase (MMP) is a member of the MMP family, which is a zinc-dependent endopeptidase that degrades all extracellular matrix components and vascular basement membrane. Survivin plays a leading role in the process of apoptosis regulation. Survivin is unique for its expression in human malignancies but not in normal adult cells. This study aimed to assess immunoexpression of MMP-2 and survivin in pancreatic carcinoma.
Patients and methods A total of 25 pancreatic carcinomas were evaluated for immunohistochemical expression of MMP-2 and survivin. The expression was evaluated by immunoreactive score that combines the intensity of immunoreactions with the percentage of positive cells.
Results In this study, 24/25 of the cases were pancreatic ductal adenocarcinoma (PDAC) and 1/25 was acinar cell carcinoma. Regarding tumor differentiation, PDAC included five cases of adenocarcinoma that were well differentiated, 16 cases were moderately differentiated, and three cases were poorly differentiated. Pancreatic carcinoma specimens showed positive cytoplasmic MMP-2 staining with high expression in 22/25 (88%) studied specimens ranging from 6/9 to 9/9 with high extent and intensity. Survivin staining with high expression was seen in 23/25 (92%) studied specimens, ranging from 9/12 to 12/12 with high extent and intensity.
Conclusion MMP-2 and survivin expressions were positive in pancreatic carcinoma with various grades but negative in non-neoplastic parenchymal, acinar, and pancreatic ductal epithelium. There were significant positive relationships between MMP-2 and survivin expression and poor histological grade in PDAC (P<0.024 and 0.021, respectively).
Keywords: matrix metalloproteinases, pancreatic carcinoma, survivin
|How to cite this article:|
Mohamed SA, Eldowik YM. Expression of matrix metalloproteinase-2 and survivin in pancreatic carcinoma: an immunohistochemical study. Al-Azhar Assiut Med J 2022;20:280-6
|How to cite this URL:|
Mohamed SA, Eldowik YM. Expression of matrix metalloproteinase-2 and survivin in pancreatic carcinoma: an immunohistochemical study. Al-Azhar Assiut Med J [serial online] 2022 [cited 2023 Jan 27];20:280-6. Available from: http://www.azmj.eg.net/text.asp?2022/20/3/280/358031
| Introduction|| |
Pancreatic carcinoma is one of the few carcinomas that has increasing incidence. Compared with other cancers, pancreatic carcinoma has the least improvement in survival over the past three decades with 5-year survival rate of only 6% . Pancreatic ductal adenocarcinoma (PDAC) has become equivalent to ‘pancreatic cancer’ and is considered the most important and most common pancreatic cancer that has rapid mortality and also as the third leading cause of cancer-related deaths in the United States . Actually, early diagnosis and radical surgery are considered the only chance of long-term survival for patients with pancreatic carcinoma. Although there has been an improvement in the management, still after effective treatment of pancreatic carcinoma, the 5-year survival rate for the patients is only 25–35% . Local invasion and distant metastasis are important biological characteristics for carcinoma of the pancreas, causing the decrease of chance for surgery and patient death. The local invasion and metastasis are a complex mechanism implicated in a set of multistep processes in the cell, among which extracellular matrix proteolytic degradation is an essential step . Matrix metalloproteinase-2 (MMP-2) is a member of the MMP family, which has an important role in the growth of some neoplastic diseases . MMP-2 is a zinc-dependent endopeptidase that degrades all extracellular matrix components and basement membrane of blood vessels. The MMP family plays an important part in the development of numerous neoplasms and connective tissue diseases. They are closely involved in invasion and metastasis of the tumor . Survivin plays an important role in the regulation of apoptosis. Survivin is distinctive for its expression in human cancers but not in normal cells. It has been involved in chemotherapy sensitization and also as a prognostic marker in several human malignancies . Immunohistochemical expression of survivin is associated with metastasis, local recurrence, and poor prognosis .
This study aimed to assess immunoexpression of MMP-2 and survivin in pancreatic carcinoma.
| Patients and methods|| |
A total of 25 formalin-fixed paraffin-embedded tissue blocks of pancreatic carcinoma and another five tissue blocks of chronic pancreatitis as a control group were retrieved for this study from the archived material of Pathology Laboratory, Sohag University Hospitals, and Sohag Oncology Center during the period from 2015 to 2019. Ethical approval to perform this work was obtained from the Institutional Ethical Committee. Data regarding patients’ age and sex, tumor size, shape, and status of distant metastasis were obtained from patients’ clinical files. Four-micrometer tissue sections were prepared from formalin-fixed paraffin-embedded tissue blocks and then stained by routine hematoxylin and eosin. The tumors were reviewed for tumor histological type, grade, perineural invasion, lymphovascular, vascular invasions, and presence of regional lymph node metastasis. Tumor grade and pathological stage were evaluated according to WHO recommendations . Immunostaining using the technique of the peroxidase-labeled streptavidin biotin to detect MMP-2 and survivin expressions was done. Five-micrometer-thick tissue sections mounted on Poly-Lysine-coated slides were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked using peroxidase-blocking reagent (Cat # TP-015-H; LabVision Corporation, 47777 Warm Springs Blvd. Fremont, CA 94539 USA). The antigen sites were unmasked by immersing the slides in sufficient amounts of 10-mmol sodium citrate buffer, pH 6.0. Sections were incubated in a microwave for 15 min, allowed to cool down, and then washed in distilled water and then in phosphate-buffered saline (PBS, pH 6.0). Sections were incubated in normal goat serum to block nonspecific interactions. Tissue sections were incubated overnight at normal temperature with MMP-2 rabbit polyclonal antibody (25 μm, concentrate, Cat#42599; GeneTex Corporation, 47777 Warm Springs Blvd. Fremont, CA 94539 USA) at a dilution of 1/100 and survivin rabbit polyclonal antibody (Cat#)RB-9245-P0; Thermo Scientific, 47777 Warm Springs Blvd. Fremont, CA 94539 USA) at a dilution of 1/50, and then resulting immune complex was detected by a universal staining kit. Tissue sections were treated with biotinylated polyvalent goat anti-mouse secondary antibody (LabVision Corporation), and then peroxidase-labeled streptavidin was applied at room temperature for 15 min, rinsed. Then, sections were counterstained in hematoxylin, washed, dehydrated, and cleared.
Positive and negative controls
Tissue sections from carcinoma of the breast were used as a positive control for MMP-2 antibody. Negative control sections were prepared from pancreatic carcinoma, but by adding PBS instead of the primary antibody. Tissue sections from colonic carcinoma were used as a positive control for survivin antibody. Negative control sections were prepared from pancreatic carcinoma, but by adding PBS instead of the primary antibody.
Evaluation of matrix metalloproteinase-2 and survivin protein expression
MMP-2 protein expression was assessed according to the percentage and the intensity of stain in the cytoplasm of cells. The stained cell percentages were divided as follows: 0 for absent staining, one point for less than 10%, two points for 10–30%, and three points for more than 30% of stained cells. The intensity of staining was divided as follows: 0 (absent), 1 (weak), 2 (moderate), and 3 (strong). The immunoreactive score (IRS) for each case was obtained by multiplication of percentage and intensity of staining. A final total score less than or equal to 3 was considered as low expression and a score of 3 was considered to be high expression . Survivin protein expression was assessed according to the percentage of positive tumor cells in five high power fields (×400) divided into five categories: (1) 0= less than 5%, (2) 1=5–25%, (3) 2=25–50%, (4) 3=50–75%, and (5) 4= more than 75%. The intensity of survivin cytoplasmic immunostaining was evaluated as follows: (a) weak=1+; (b) moderate=2+; and (c) intense=3+. The IRS for each case was obtained by multiplication of percentage and intensity of staining .
Data entry and analysis of data were done using SPSS, version 20 (Statistical Package for Social Science, Philadelphia, PA, USA). Data were presented as number, percentage, mean±SD, and median. The correlations between survivin and MMP-2 expression and other clinicopathological data were determined using a Mann–Whitney test for quantitative variables and a Kruskal–Wallis test for qualitative variables. To measure the correlation between quantitative variables, Spearman correlation was done. P value was considered significant if P value less than 0.05.
| Results|| |
Clinical data and pathological characteristics of the studied specimens
The age range of the 25 patients with pancreatic carcinoma ranged from 40 to 69 years old, with a mean±SD of 56.32±8.57 years, and the median age was 59 years old. Patients with pancreatic carcinoma included in this study comprised 14 males and 11 females. More than half of the patients with pancreatic carcinoma (56%) were clustered in the age group less than 60 years old. The size of tumor masses ranged from 1 to 7 cm (six specimens ≤2 cm and 19 specimens >2 cm). Specimens were taken by either Whipple operation (pancreaticoduodenectomy) or distal pancreatectomy. Most of these tumor masses (18/25) were located at the pancreatic head and the rest (7/25) were located at the pancreatic body and tail.
On histopathological typing, 24/25 cases of the specimens were PDAC and 1/25 was acinar cell carcinoma. Regarding tumor differentiation, PDAC included 5/24 cases of well-differentiated adenocarcinoma, 16/24 cases of moderately differentiated, and 3/24 cases of poorly differentiated. According to the WHO staging system of pancreatic carcinoma , the studied cases have been classified pathologically into pT1, pT2, pT3, and pT4 in six, 12, six, and one case, respectively.
Pancreatic carcinoma cases showed positive cytoplasmic MMP-2 staining with high expression in 22/25 (88%) studied specimens. Non-neoplastic acinar parenchymal and pancreatic ductal epithelium was negative in all 25 cases in this study. Only 3/25 (12%) of the studied specimens showed positive MMP-2 staining with low MMP-2 expression. Of five well differentiated, four cases showed high MMP-2 expression and one case showed low expression. Of 16 moderately differentiated PDACs, 14 cases showed high MMP-2 expression and two cases showed low expression. All three poorly differentiated PDACs showed high MMP-2 expression. The acinar cell carcinoma case showed high MMP-2 expression. Poorly differentiated PDAC showed high MMP-2 expression in 3/3 specimens. A specimen of acinar cell carcinoma showed high MMP-2 expression. The surrounding normal pancreatic tissue and five specimens of chronic pancreatitis showed negative MMP-2 staining. Stromal cells showed focal positive MMP-2 staining in 12/25 of specimens ([Table 1], [Figure 1], Graph 1 and 2). Regarding survivin staining, there was high expression in 23/25 (92%) studied cases. Non-neoplastic acinar parenchymal and pancreatic ductal epithelium was negative in all 25 cases. Only 2/25 (8%) of pancreatic carcinoma cases showed low survivin expression. Of five well-differentiated PDACs, four cases showed high survivin expression and one case was of low expression. Of 16 moderately differentiated PDACs, 15 cases showed high survivin expression and one case showed low expression. All three poorly differentiated PDACs showed high survivin expression. The only case of acinar cell carcinoma showed high survivin expression. A specimen of acinar cell carcinoma showed high survivin expression. The surrounding normal pancreatic tissue and stromal cells showed negative staining for survivin ([Table 2], [Figure 2], Graph 3 and 4). There were significant relationships (P<0.024) between MMP-2 and survivin expressions and tumor grade in PDAC. There were no significant relationships between MMP-2 and survivin expressions and other studied parameters.
|Table 1 The relationship between matrix metalloproteinase-2 expression and some clinicopathological parameters|
Click here to view
|Figure 1 MMP-2 expression in different histological types of pancreatic carcinoma (avidin-biotin). Cytoplasmic expression of MMP-2 in PDAC: (a) well differentiated, (b) moderately differentiated, (c) poorly differentiated, and (d) acinar cell carcinoma of pancreas. Immunostained section, magnification: ×400 for all. MMP-2, matrix metalloproteinase-2; PDAC, pancreatic ductal adenocarcinoma.|
Click here to view
|Figure 2 Survivin expression at different histological types of pancreatic carcinoma (avidin-biotin). Cytoplasmic expression of survivin in PDAC: (a) well differentiated, (b) moderately differentiated, (c) poorly differentiated, and (d) acinar cell carcinoma of pancreas. Immunostained section, magnification: ×400 for all. PDAC, pancreatic ductal adenocarcinoma.|
Click here to view
|Table 2 Relationship of survivin expression and some clinicopathological parameters|
Click here to view
| Discussion|| |
The current study was carried out on 25 formalin-fixed paraffin-embedded tissue blocks of pancreatic carcinoma cases and five cases of chronic pancreatitis as a control. The mean age of the cases was 56.32 years. Males were more affected with pancreatic carcinoma than females.
Histopathological examination showed that 24 specimens were PDAC; 5/25 cases of well differentiated, 16/25 cases of moderately differentiated, and 3/25 cases of poorly differentiated pancreatic carcinoma, and one specimen was acinar cell carcinoma. Perineural invasion was observed in 20/25, whereas vascular invasion was observed only in 2/25 of the studied tumors. Involvement of lymph node was observed in 13/25 of the tumors. Regarding tumor stage, most cases of this study were classified as either pT2, with 12/25 cases, or pT3, with 6/25 cases.
In this study, we investigated the MMP-2 and survivin expression and examined their relationships with the clinicopathological features of patients with pancreatic carcinoma. Immunohistochemical-MMP-2 expression was cytoplasmic in pancreatic carcinoma cells, and its level is generally high as the IRS was 9/9 or 6/9 with high extent and intensity. There expression was detected in 22/25 (88%) and 23/25 (92%) cases of pancreatic carcinomas, respectively. In this study, a significant relationship was found between expression of MMP-2 in cases of pancreatic carcinoma and tumor grade (P<0.024). This finding was nearly similar to a previous study , which found a significant correlation between expression of MMP-2 in pancreatic carcinoma and grades of tumor differentiation. Moreover, others  reported that expression of MMP-2 expression in carcinoma of pancreas was significantly correlated with poor histological grade. There was no significant relation between MMP-2 expression and other studied clinicopathological parameters of pancreatic carcinoma: tumor size, tumor stage, lymphovascular invasion, lymph node metastasis, or perineural invasion. However, one study  found only a significant correlation with female sex, whereas another study found a significant correlation with patients’ age . Some studies demonstrated the prognostic significance of expression of MMP-2 in many types of tumors Some reports have shown that  MMP-2 overexpression was significantly associated with a decreased survival rate and being a prognostic marker for progression of bladder cancer.
Regarding survivin expression in pancreatic carcinoma, our results were similar to those reported by Lee et al. , who demonstrated that survivin expression in pancreatic carcinoma was positive in 93.9%. Another study  reported that 88% of cases were positive for survivin, and Satoh et al.  reported that 77% of pancreatic carcinoma cases were positive for survivin. In the current study, there was a significant relationship between survivin immunoexpression in cases of pancreatic carcinoma and tumor grade (P<0.021). This finding was nearly similar to that reported by others , who found a significant correlation between expression of survivin in pancreatic carcinoma cases and grades of tumor differentiation. There was no statistically significant relation between expression of survivin and other studied clinicopathological parameters such as lymph node metastasis or tumor stage. Satoh et al  also reported no statistically significant relation between the degree of survivin immunostaining and grade and stage of PDAC. In contrast, Qiao et al. , reported that there is a positive correlation between survivin immunostaining and TNM staging. Absence of correlation between expression of survivin and most of the clinicopathological parameters of pancreatic carcinoma in the current study, which may be owing to a small sample size. All studied cases of pancreatic carcinoma with advanced stages (T3/T4) showed high survivin expression, and 88.9% cases with less advanced stages (T1/T2) showed high survivin expression but with no significant relation. Survivin was expressed in most pancreatic carcinoma cases, and the extent of its expression strongly correlated with the aggressiveness of malignant cells and poor prognosis. These results elevate many exciting diagnostic and therapeutic possibilities. First, inhibition of survivin immunoexpression by anti-sense treatment has been reported to elevate the chemotherapy effectiveness  and also affect radiotherapy, especially in light of experimental evidence that survivin acts as a radio-resistance factor in carcinomas of pancreas .
| Conclusion|| |
MMP-2 and survivin immunoexpressions were positive in pancreatic carcinoma with various grades but negative in non-neoplastic parenchymal, acinar, and pancreatic ductal epithelium. There were significant correlations between MMP-2 and survivin expressions and poor histological grade in PDACs (P<0.024 and 0.021, respectively), but not with other clinicopathologic parameters. So, the need to perform further studies on a larger number of cases is advised.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Siegel R, Ma JM, Zou ZH, Jemal A. How early can pancreatic cancer be recognized? a case report and review of literature: cancer statistics. Cancer J Clin 2014; 64:9–29.
Mostafa ME, Ipek ES, Burcin P, Burcin P, Volkan A. Pathologic classification of pancreatic cancers. Chin Clin Oncol 2017; 6:59.
Mitchem JB, Hamilton N, Gao F, Hawkins WG, Linehan DC, Strasberg SM. Long-term results of resection of adenocarcinoma of the body and tail of the pancreas using radical antegrade modular pancreatosplenectomy procedure. J Am Coll Surg 2012; 214:46–52.
Binker MG, Binker-Cosen MJ, Binker-Cosen AA, Binker-Cosen LI. Microenvironmental factors and extracellular matrix degradation in pancreatic cancer. JOP 2014; 15:280–285.
Roy R, Zurakowski D, Wischhusen J, Frauenh Offer C, Kulke M, Moses MA. Urinary TIMP-1 and MMP-2 levels detect the presence of pancreatic malignancies. Br J Cancer 2014; 111:1772–1779.
Zhai LL, Cai CY, Wu Y, T ZG. Correlation and prognostic significance of MMP-2 and TFPI-2 differential expression in pancreatic carcinoma. Int J Clin Exp Pathol 2015; 8:682–691.
Sarela AI, Verbeke CS, Ramsdale J, Davies CL, Markham AF, Guillou PJ. Survivin in pancreatic cancers. Br J Cancer 2002; 86:886–892.
Goossens-Beumer J, Zeestraten ECM, Benard A, Christen ET, Reimers MS, Keijzer R, Sier CFM. Combined Aldh1, survivin, and EpCAM predict survival. Br J Cancer 2014; 29:35–44.
Cong L, Liu Q, Zhang R, Cui M, Zhang X, Gao X et al.
Tumor size classification of the 8th edition of TNM staging system is superior to that of the 7th edition in predicting the survival outcome of pancreatic cancer patients after radical resection and adjuvant chemotherapy. Sci Rep 2018; 8:10383.
Jakubowska K, Pryczynicz A, Januszewska J, Sidorkiewicz I, Kemona A, Niewiński A et al.
The expression of matrix metalloproteinases-2 and -9 and their tissue inhibitor 2 in pancreatic ductal and ampullary carcinoma and their relation to angiogenesis and clinicopathological parameters. Anticancer Res 2008; 28:1875–1882.
Jakubowska K, Pryczynicz A, Januszewska J, Sidorkiewicz I, Kemona A, Niewiński A et al.
Expressions of matrix metalloproteinases 2, 7, and 9 in carcinogenesis of pancreatic ductal adeno-carcinoma. Dis Markers 2016; 2016:9895721.
Vasala K, Pääkkö P, Turpeenniemi-Hujanen T. Matrix metalloproteinase-2 immunoreactive protein as a prognostic marker in bladder cancer. Urology 2003; 62:952–957.
Lee MA, Park G, Lee H, Lee H-J, Jin-Hyoung K, Kang H-J et al.
Survivin expression and its clinical significance in pancreatic cancer. BMC Cancer 2005; 5:127.
Satoh K, Kaneko K, Hirota M, Masamune A, Satoh A, Shemosegawa T et al.
Survivin in pancreatic tumors. Am Cancer Soc 2001; 2:271–276.
Qiao JG, Zhang YQ, Yin YC, Qiao JG, Yin Y-C, Tan Z et al.
Survivin and Bcl-2 in pancreatic cancer. World J Gastroenterol 2004; 10:18.
Olie RA, Simoes-Wust AP, Baumann B, Robert A, Olie A, Baumann B et al.
A novel antisense nucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells tochemotherapy. Cancer Res 2000; 60:2805–2809.
Asanuma K, Moriai R, Yajima T, Yagihashi A, Yamada M, Kobayashi D, Watanabe N. Survivin as a radioresistance factor in pancreaticcancer. Jpn J Cancer Res 2000; 91:1204–1209.
[Figure 1], [Figure 2]
[Table 1], [Table 2]