ENHANCER OF ZESTE HOMOLOG 2 AND VASCULAR ENDOTHELIAL GROWTH FACTOR IMMUNOHISTOCHEMICAL EXPRESSION ARE ASSOCIATED WITH ADVERSE CLINICAL OUTCOME IN NON-METASTATIC CLEAR CELL RENAL CELL CARCINOMA

Mona Mostafa Ahmed 1 , Mai M. abdelwahab 1 , Safa A. Balata 2 and Hassan R Ashour 3 . 1. Department of pathology, Faculty of Medicine, Zagazig University, Egypt. 2. Department of Medical Oncology, Faculty of Medicine, Zagazig University, Egypt. 3. Department of General surgery, Faculty of Medicine, Zagazig University, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

. Von-Hippel Lindau gene inactivation is common in both hereditary and sporadic renal cell carcinomas resulting in hyperactivity of the hypoxia-inducible factor leading to production of factors that stimulates blood vessel formation as vascular endothelial growth factor. VEGF is a potent angiogenic factor that induces angiogenesis during tumorigenesis (Lainakis and Bamias, 2008). Ki-67 is a protein that present in all the phases of cell cycle (G1, S, G2 and mitosis) while it is absent in non-dividing cells (G0) (Gerdes et al., 1984).That is why it is considered as excellent indicator for the proliferating fraction of tumor cells. Instead of relying on multiple parameters, Ki-67 needs only evaluation of nuclear staining for interpretation. Proliferation determined by Ki-67 is known to be of prognostic importance in CCRCC and is known to correlate with tumor grade (Mehdi et al., 2016). The aim of the study is to assess the significance of immunohistochemical expression of EZ2H and VEGF on tumor behavior, prognosis and patient survival in clear renal cell carcinoma.

Patients And Methods:-
This study was conducted in Pathology, Clinical oncology and General surgery departments, Zagazig University, in the period from January 2015 to February 2017. The study was carried out on 25 patients of clear cell renal cell carcinoma (CCRCC). Sections from the involved cases were stained, evaluated by routine H&E stain and were graded according to Fuhrman grading system (Fuhrman et al., 1982),and the stage is evaluated conferring to the American Joint Committee on Cancer staging system(Edge and Compton, 2010).Clinical, radiological and pathological data were abstracted from files of the corresponding departments. Patients with a negative postoperative PET/CT scan were included in this study while those with evidence of metastasis or deficient data were omitted. None of the patients had received chemo or radiotherapy preceding surgery. Clinical follow-up was done every three months to all cases and information concerning follow up was abstracted from hospital records or patient contact.

Immunohistochemistry:-
The immunohistochemical staining procedure was done using streptavidin-biotin immunoperoxidase technique (Dako-Cytomation, Glostrup, Denmark). Sections of 3-5 µm from the formalin-fixed-paraffin-embedded blocks were cut and mounted on positively charged slides then deparaffinized by xylene, and rehydrated in graded alcohol. Thereafter, sections were boiled in buffered citrate (pH 6.0) for about 20 minutes then washed in PBS (pH 7.3). Then, endogenous peroxidase activity was blocked with 6% H2O2 in methanol. The slides were incubated overnight with a mouse monoclonal EZH2 (ab14389; Abcam, Cambridge, UK, 1:100); rabbit polyclonal VEGF (Lab Vision, Fremont, CA, USA, 1:200) and mouse monoclonal Ki-67 (Lab Vision, Fremont, CA, USA, 1:50) antibodies. After rinsing in PBS, the slides were immersed with a biotin-conjugated secondary antibody (Lab Vision Corporation, Fermont, USA). DAB was used as a chromogen and Mayer's Hematoxylin was used as a counter stain, and then the slides were washed with distilled water and PBS. Positive and negative controls were stained with the same setting of the studied cases. Sections from normal testis were used as positive controls for EZH2 and prostate adenocarcinoma tissues as positive controls for VEGF and Ki-67. The negative controls were done using the same tissue with the omission of the primary antibody.
Statistical Analysis:-Continuous variables were expressed as the mean ± SD & median (range), and the categorical variables were expressed as a number (percentage). Continuous variables were checked for normality by using Shapiro-Wilk test. Independent samples Student's t-test was used to compare between two groups of normally distributed variables. Kruskal Wallis H test was used to compare between more than two groups of non-normally distributed variables. Percent of categorical variables were compared using Pearson's Chi-square test or Fisher's exact test when was appropriate. Strength of relationship between immunohistochemical staining for EZH2, VEGF and Ki-67 were examined using computing Kendall's tau-c correleation coefficient, (+) sign was indicator for direct relationship & (-) sign was indicator for inverse relationship, also values near to 1 was indicator for strong relationship & values near 0 was indicator for weak relationship.
Disease free survival (DFS) was calculated as the time from surgery to reappearance of the disease (local or regional or distant metastasis) or the most recent follow-up in which relapse free. Overall Survival (OS) was calculated as the time from diagnosis to death or the most recent follow-up contact (censored). Stratification of DFS and OS was done according to clinicopathological data and markers score. These time-to-event distributions were estimated using the method of Kaplan-Meier plot, and compared using two-sided exact log-rank test. A p-value <0.05 was considered significant. All statistics were performed using SPSS 22.0 for windows (SPSS Inc., Chicago, IL, USA) and MedCalc windows (MedCalc Software bvba 13, Ostend, Belgium).
Immunohistochemical results:-EZH2 immunohistochemical results:-EZH2 was expressed in 88% of our studied cases. There was a statistically significant correlation between its expression and tumor grade ,where most of low grade tumors (GI,II) showed low scores (1and 2),and none of them showed high scores, in contrast to high grade tumors (GII,VI) that showed only high scores (3 and 4)(p< 0.001). As regards the relationship between EZH2 and tumor size, there was a gradual increase in its expression with increasing tumor size ,as 6.7% and 25% of T1 and T2 tumors showed score 4 compared to 80% and 100% of T3 and T4 respectively, with a significant statistical difference (p< 0.001).EZH2 was also correlated with nodal metastasis (p< 0.0016).
The relationship between EZH2 score and tumor stage was statistically significant as none of stage I or II tumors showed score 4 ,but 77.8% of stage III tumors showed this score(p< 0.001). However, no significant difference was detected between EZH2 and age or sex of the patient (p= 0.771 & 0.417) respectively (Table 2, Figure 1).

VEGF immunohistochemical results:-
VEGF showed low expression in 48% and high expression in 52% of the studied cases. A significant association was observed between VEGF expression and tumor grade, as all grade 1 cases showed low expression ,while all grade III and VI cases showed high expression(p< 0.001). VEGF expression was also correlated with tumor size (p=0.001) and tumor stage,99.3% of stage I showed low expression, while all cases of stage II and III showed high expression(p< 0.001). However, no significant correlation was observed between VEGF and nodal metastasis (p=0.09), age (p= 0.56) or sex (p= 1.00)of the patients (Table 3,Figure2). 1311 Correlation between EZH2, VEGF and Ki-67 immunohistochemical expressions: Using Kendall's tau-b correleation coefficient, a significant correlation was detected between EZH2 and VEGF expression (τ tau correleation coefficient = +0.941 ,p< 0.001), and a significant positive correlation was detected between ki-67 and both EZH2 ( τ tau correleation coefficient =+0.414,p= 0.003), and VEGF (τ tau correleation coefficient =+0.602 ,p<0.001) (Table 4, Figure 1

,2,3)
Survival analysis results:-During 24 months of follow up, fourteen patients relapsed. Disease Free Survival (DFS) was considered from the time of surgery to confirmation of locoregional recurrence (LRR) or distant metastasis (DM), either radiological or clinical, or death by any cause and patients were censored at the last time known to be disease free and alive. Overall Survival (OS) was calculated from the time of surgery to death from any cause and patients were censored at the date last known to be alive.
Cases with high EZH2 expression were significantly associated with disease progression (both loco-regional recurrence and distant metastasis), disease free survival and a poor overall survival (p< 0.001). The expression of VEGF was significantly associated with the progression of disease (both loco-regional recurrence and distant metastasis), disease free survival and a poor overall survival (p< 0.001, p< 0.001and p= 0.009 respectively)(Table 5&6 , Figure 4).         In this study, significant direct correlation between EZH2 expression and Fuhrman grade (p< 0.001) that acts as independent prognostic factor for CCRCC was detected. Low EZH2 expression was noticed in lower grades renal cancers, while high expression was noticed in higher grades renal cancers. These results were in line with that of   ., 2003).The loss of miRNAs such as miR-26a, miR-101 and miR-214 lead also to EZH2 accumulation (Danget al.,2012).Treatment with EZH2 inhibitor drugs as 3-deazaneplanocin A33, or blocking the effect of EZH2 may provide a major advance in the treatment of CCRCC (Crea et al., 2012).
Ki-67 has an independent prognostic importance in renal cell carcinoma, and also correlates with Fuhrman nuclear grade but is more objective and reproducible and can be used in conjunction with it to determine prognosis in renal cell carcinoma (Mehdi et al., 2016).
Significant correlation between Ki-67 and EZH2 expression (p= 0.003) was found. As far as we know, no previous studies tested the relation between EZH2 and Ki67 expression in CCRCC. According to current work, EZH2 is a negative prognostic marker in patients of CCRCC; that is why the assessment of its expression may improve selection of patient for systemic therapies. EZH2 status integration into current prognostic models may improve survival prediction.
Vascular endothelial growth factor (VEGF) is considered as the most potent endothelial cell-specific angiogenesis factor. It increases vascular permeability leading to endothelial cell proliferation with subsequent tube formation. In addition, CCRCC, a clinically angiogenic activity, has a direct relation with the expression of VEGF. This led to VEGF inhibition-based treatment methods used today against CCRCC ( In this work, VEGF expression in CCRCC was significantly related to more progressive disease in correlation with lower survival rates and this in line with results of Osman and Youssef (2015).

Conclusion:-
Compared with early staged tumors, advanced CCRCC had significant detectable EZH2 and VEGF expressions. The expression level of EZH2 correlated positively with that of VEGF in CCRCC. The present study suggests that EZH2 overexpression has a role in the progression, development and angiogenesis in renal cell carcinoma and consequently can be used as an indicator for predicting the prognosis of CCRCC patients. These results suggest that EZH2 targeting might be an attractive therapeutic approach in the treatment of renal cancer.

Conflict of interest:
There are no conflicts of interest.