Correlation of Survivin and MMP 9 Expressions with Prognosis and Clinicopathological Parameters in Surface Epithelial Ovarian Carcinomas

Objective: Pathological stage is the most important prognostic factor in ovarian carcinomas, and many studies have been carried out to predict prognosis. In our study, we searched the expression of survivin, which prevents apoptosis and matrix metalloproteinase-9 (mmP-9), which promotes the metastasis of the tumour by splitting up the basal membrane in primary epithelial ovarian carcinomas. We aimed to observe their effects on prognosis by comparing clinicopathological parameters.


InTRoduCTIon
Ovarian cancer is the most lethal gyneacological malignancy and ranks 5th in cancer-related women deaths in the USA (1).In spite of advances in treatment during the last 20-30 years, there has been no significant decrease in death rate caused by the ovarian cancer and the average 5-year life span has been limited to % 10-30 (2)(3)(4)(5).At present, pathological stage is the most important prognostic factor known in ovarian tumours (6)(7)(8).
Numerous markers have been studied with immunohistochemical or cytogenetic methods in order to explore a prognostic and predictive factor in surface epithelial ovarian carcinomas.Survivin and matrix metalloproteinase-9 (mmP-9) are also among the most overemphasized markers in this issue (9,10).
In recent studies, survivin was proven to play a vital role in the invasion and metastases of ovarian epithelial carcinomas (3,11).Survivin (BIrC5: Baculoviral inhibitor of apoptosis repeat-containing 5) belongs to the gene family that inhibits apoptosis and it prevents the apoptosisrelated death of cancer cells (3,11,12,13).While it is not present in many normal cells, its distinct expression in cancer cells is considered an indicator of poor prognosis (9,(12)(13)(14)(15)(16)(17)(18).Ovarian tumours, with cytoplasmic survivin positivity, were reported to be more resistant to taxol/ platinum treatment (19).
The tumour cells which escape from apoptosis spread to the surface of the ovarian capsule and break up the basal membrane with mmP.This step is an important stage in terms of tumour progression (3,20).mmPs are homologue enzyme family that includes zinc in its active region and has the ability to break up extracellular matrix (ECm) and basal membrane components (21,22).They also take part in pathological processes like tumour cell invasion, angiogenesis and metastasis (21,22).The expression of mmP-9 was detected at a higher rate in ovarian cancers compared to the normal ovarian tissue, benign and borderline tumours (23).Furthermore, its expression was demonstrated in many metastatic ovarian carcinomas (21,24).mmP-9 is expressed in both epithelial and stromal cells of ovarian tumours (23,24,25,26).Both stainings have been associated with poor prognosis (24,26).Particularly stromal staining has been associated with advanced stage, development of metastasis, the presence of ascites and shorter survival times compared to the tumours without stromal expression (23)(24)(25)(26)(27).
The purpose of this study is to evaluate the expressions of survivin and mmP-9 with clinicopathological parameters in surface epithelial ovarian carcinomas and to detect their effects on prognosis.

MATeRIAl and MeThod
This study consisted of sixty patients who were diagnosed with epithelial ovarian carcinoma between 2003 and 2008, in Department of Pathology, Izmir Atatürk Training and research Hospital and had been treated at Department of medical Oncology of the same hospital and followed-up for at least 1 year.Patients' age, tumour's diameter, localisation, histological type, grade, stage, recurrence, metastatic conditions and the survival data were obtained from the archive records.
The Gyneacologic Oncology Group (GOG) grading system was used in grading the tumours that belonged to the cases and the International Federation of Gyneacology and Obstetrics (FIGO) was used in staging the tumours.The terms disease-free survival (DFS) and overall survival (OS) were used for prognostic evaluation of the patients.DFS was defined as the time from the diagnosis to the disease recurrence or the last visit which came first for early-stage (stage I-II) patients.On the other hand OS was defined as the time from diagnosis to the last visit or death, whichever came first.
Immunohistochemical staining was performed on formalinfixed and parafin embedded tissue using the Streptavidinbiotin complex method.Tissue sections were incubated with primary antibodies anti Survivin (monoclonal mouse anti-human Survivin Clone 12C4, Dako Denmark) 1/100 dilution; mmP-9 (mmP-9, 92 kDa Collagenase IV, Epitope Specific rabbit Antibody, Spring Biosence) ready to be used was applied for 2 h at room temperature.Normal testis and placenta tissues were used as positive control for survivin and mmP-9 staining respectively.
For immunohistochemical analysis, all the tumour areas were examined at low magnification (x10).For each antibody in the epithelial cells, at x20 magnification, the percantage of stained tumour cells, and at high magnification (x40), the intensity of tumour cells were determined semiquantitatively and subjectively.Immunohistochemical evaluation was performed by modifying the method used in the study of Kamat et al (24).For survivin and mmP-9, cytoplasmic staining in epithelial cells was taken into consideration and the total score was obtained by adding up the values given to staining percentage and intensity.Accordingly, for staining percentage, 0-5%=0, 6-50%=1, 50%>2; for staining intensity, weak=1, moderate=2, strong=3 points were given.Overall score was obtained by adding up these points and the following scores was defined: 0= negative   III).moreover, no significant effect on disease free survival and overall survival was observed as well.
Epithelial staining in mmP-9 was compared with all clinicopathologic parameters like survivin (Table IV).
The only significant correlation was detected between the staining intensity of mmP-9 and histological grade (p=0.028).As tumour grade increase, mmP-9 staining intensity increase as well.Stromal staining with mmP-9 Statistically; histological type, tumour's grade, tumour diameter, stage and age were compared with each other.However, no statistical association was obtained from this comparison.Additionally the effects of these parameters on survival were investigated.Distant metastases rate was significantly higher in advanced stages (p=0.005)and overall survival of the patients with advanced-stage disease was significantly shorter than that of early stage patients (11 months vs 34 months; 95% CI; p=0.0269).As expected, these results supported the fact that in advanced stage patients, risk of recurrence and death rates increase when OS period decreases.

dISCuSSIon
In this study, all clinicopathological parameters were analysed.Consistent with previous studies, recurrence risk and death rate was higher and overall survival was shorter in advanced-staged patients when compared with patients with early stage disease (7,8,24).
Similar with the results of the previous studies, survivin expression did not have any relationship with stage and histological type in our study (9,28).moreover, in contrast with previous reports, survivin expression was not associated with tumour grade and disease prognosis (9,28,29,30,31).This result may be due to the fact that the majority of tumours in our study group were of moderate or high grade.Similarly, no association was detected between survivin expressions and the parameters such as tumour diameter, recurrence and survival.The data obtained from the previous studies suggest that survivin expression shortens the OS and DFS periods (28,29,30,32).In this study, no statistically significant result could be obtained between survivin OS and DFS periods same as study by Cohen et al. (9).
In our study, mmP-9 expression in epithelial cells was compared with parameters like stage, histological type, and tumour diameter.No statistical significance was present, which was in paralel with the findings in most studies (24,33,34).However the correlation between the intensity of mmP-9 in epithelial cells and histological grade was statistically significant which was similar with the study by Sillanpää et al. (25).
Similar to epithelial mmP-9 expression, no significant relationship was observed between the stromal expression of mmP-9 and all the clinicopathological parameters as well.Kamat et al. demonstrated that stromal expression of mmP-9 was associated with advanced-staged and high-grade tumours (24).Additionally, they showed that lymph node involvement, the presence of ascites, serous morphology and suboptimal cytoreduction rates were more common in tumors with stromal expression of mmP-9.Sillanpää et al. established a significant correlation between high stromal positivity and advanced stage while they could not detect any relationship between histological grade, histological type and patient's age (25).In present study, similar to previous studies, statistically significant results were observed between mmP-9 stromal staining values and DFS times (24,25).However, the correlation between stromal staining and OS period was insignificant.Ozalp et al. detected a correlation between stromal staining and shortened OS period (34).In our study, the reason for was negative in 31 cases and positive in 29 cases (Figure 3).mmP-9 stromal staining values were compared with parameters such as disease stage, grade, histological type and tumour size; no significant difference was observed.DFS was significantly shorter in patients with stromal mmP-9 expression than the patients without expression (23 months vs 45 months; 95% CI; p=0.0326) (Figure 4).The patients with stromal mmP-9 expression had relapsed significantly earlier than the patients without stromal mmP-9 expression.However, the same result was was not observed on OS analysis, and no statistically OS difference was observed in patients with and without stromal mmP-9 expression.the statistically insignificant association between stromal staining and OS could be the notable differences in patients' follow-up periods.
In summary, this study demonstrates the following results: • Survivin expression was not associated with clinicopathological parameters and prognosis in this study.
• The correlation between mmP-9 staining in epithelial cells and histologic grade was significant, however, no significant impact on prognosis and OS could be demonstrated.
• Stromal expression of mmP-9 had a significantly negative effect on recurrence times, however the same effect on overall survival times could not be established.
When literature is reviewed, regarding the results obtained from survivin and mmP-9, the remarkable point is; different results had been achieved in various studies carried out on this subject and there has been no consensus yet (9,24,25,27,(28)(29)(30)(31)(32).The reasons for these contradictions might be the differences in technical usage, or differences between the antibodies, manufacturing company, clonality, incubation and dilution or several methods used in evaluation (3,9,28,32,35).Furthermore, it was emphasized that, to obtain statistically significant results, studies have to be carried out on a broad range of patient groups by using standardized methods (9,18).
Thus, in order to prove the predictive effect of the stromal expression of mmP-9 in epithelial ovarian carcinoma treatment, retrospective and prospective studies on more homogenous and broader patient groups are clearly required.

Figure 4 :
Figure 4: Kaplan-meier survival curves for DFS of epithelial ovarian cancer patients according to stromal positivity of mmP-9.
Kruskal Wallis) was used to compare the effect of a parameter between two multi-sorted variable.Survival analysis was carried out by Kaplan-meier analysis; all of the ranges were stated with a 95% confidence interval (CI).The survival comparison between two different parameters was fulfilled by using the Log-rank test.Statistical significance limit was accepted as p values under 0.05 for all tests.In demographic records, the patients' age, tumour's location, diameter, type, grade, stage, metastasis, recurrence and survival status of the patients were evaluated (TableI).Four patients had liver metastasis, four patients had pleural effusion while one patient developed pancreas and one patients lung metastasis during the follow-up period.No statistical correlation was detected in the comparison between age and stage, tumour diameter, histological type.

Table II :
Percentage, density and score distribution of Survivin and mmP-9

Table III :
Comparison of survivin staining percentage, density and scores with clinicopathological parameters