Angiopoietin-2 Immunohistochemical Expression in Oral Squamous cell Carcinoma

Background: There are various secreted proteins affecting the prognosis of oral squamous cell carcinoma (OSCC) and one of them is Angiopoietin-2(Ang-2) which is thought to have an essential role in the development and progression of the tumor. Aim of the study: This study was conducted to determine the expression of (Ang-2) in (OSCC) to assess its correlations with clinicopathological parameters of the tumor. Material and Methods: 36 formalinfixed, paraffinembedded tissue blocks histologically diagnosed as OSCC were examined for Ang-2 immunohistochemical expression semi quantitively. Results: The expression of Ang-2 was significantly associated with histopathological grade (P value=0.023), while there is no significant association with the clinical parameters analyzed in OSCC patients. Conclusion: A significant association between Ang-2 expression and histopathological grade of OSCC may predict its biological behavior.


INTRODUCTION
Oral Squamous cell carcinoma (OSCC): is a malignant epithelial tumor of oral cavity that derived from the lining stratified squamous epithelium (1) . Both genetic and environmental factors in addition to the viral infections are incorporated in the pathogenesis of OSCC (2) An important number of patients developed OSCC at early stages, and with small sized tumor, they may develop poor prognosis ,so the level of histopathological differentiation can predict the biological specific and aggressive clinical behavior of the tumor . (3;4) so when the tumor histopathologically appeared mature and look like the epithelial tissue which is originated from (quite resemblance to squamous cells, keratin pearls, less cells or nuclear pleomorphism) tend to grow slowly and not metastasized unless in latent stage which is called well differentiated, low grade, or grade Ι OSCC (5) In contrast, a tumor with marked pleomorphism and little or no keratin production may be so immature so it becomes difficult to identify the tissue of origin. graded as ΙΙΙ and called poorly differentiated or high grade OSCC. The tumor appeared in between, graded ΙΙ or so called moderately differentiated (6)  Angiopoietin-2 (Ang-2), a member of the angiopoietin family proteins functioned as ligands for the endothelial-specific tyrosine kinase receptor "Tie2" (7) .In neoplasms of different histological origin (e.g., gastric, colon, prostate, breast, and brain carcinomas),the expression of Ang-2 is elevated and linked with poor prognosis (8) .
Studies showed that Ang-2 may overexpressed in OSCC and it always associated with aggressive tumor behavior and poor prognosis (9,7) The aim of present study is to evaluate the expression of Ang-2 in number of patients diagnosed as OSCC and its association with clinicopathological parameter of the disease.

Conventional Immunohistochemistry (IHC).
After deparaffinization and rehydration were done to the prepared histological sections, blocking of endogenous peroxidase was performed by incubation them with 3 % hydrogen peroxide for 10 min. then blocking for nonspecific antibodies binding by incubation them for 1 hour with normal goat serum. Sections were then incubated with primary antibody, an anti Ang-2 monoclonal antibody (1/100) dilution; (abcam company [MM0020-1F29] ab56301) overnight at 4 o C. then washed with phosphate-buffered saline (PBS, pH = 7.0). By using the detection kit, the slides were incubated with secondary antibody (rabbit anti mouse antibody unconjugated then Gout anti -rabbit HRP conjugated) 10min for each followed by incubation with 3 , -3 ,diaminobenzidine chromogen (DAB)for 1min at 37 o C. All sections then stained with Haematoxylin as a counter stain, dehydrated through graded alcohols, and at last, mounted. For each IHC run a positive control tissue sample (normal placenta) and a negative control sample (consisted of sample in which the primary antibody were replaced by PBS) was included.

Immunohistochemical evaluation of Ang-2 antibody:
To assess the immunohistochemical expression of Ang-2, two pathologist who blinded to the any information presented in the case sheets of the patients two pathologists, who were blinded to the patients' information. Immuno-re activity of Ang-2 was primarily detected in the cytoplasm of tumor cells. The expression was performed using a semi quantitative staining intensity score: no staining was scored as 0, faint staining as 1, moderate as 2, and high intensity staining as 3 . (10) Statistical analysis Categorical variables were represented by number(n) and percentage (%) and the different percentages were tested using Chi-square test( 2 ). Statistical significance was considered whenever the P value was less than 0.05.   Table (4,5,6,7) the association between Ang-2 expression with (age group, gender, clinical presentation, sites) were found to be statistically non significance (P value>0.05).      Table (8) showed the association between Ang-2 expression and histopathological grade of OSCC of the study sample, the association was found to be statistically significant(P value=0.023).

DISCUSSION
There are numerous molecules overexpressed in OSCC, associated with aggressive behavior of the tumor and may affect its prognosis. According to the current study; Ang-2 was expressed in 75% of the cases (ranged between moderately stained as 50%, to strong stained as 25% of the cases). Although the association between the Ang-2 expression and clinicopathological parameters of the studied tumor was found to be statically non significance( P value>0.05). There was significance association between Ang-2 expression and the histological grade of the tumor (P value=0.023) which agreed with previous studies (11,7) but disagreed with ( 9,12,13) While tumor progressed, there is a great need for oxygen and the microenvironment suffered from hypoxic condition that , s stimulate ECs, tumors , cells to secret different cytokines which enhanced angiogenesis, and one of them is Ang-2 (14,15) Overexpression of Ang-2 could lead to decrease cell apoptosis so promotes tumorgenesis, on the other hand, Ang-2 thought to have an essential role in the angiogenesis process by enhanced epithelial-mesenchymal transition " ( Previous studies showed that Ang-2 induces transformation of noncancerous liver to hepatocellular carcinoma (19) . In addition, that Ang-2 mostly associated with disease progression, metastasis, and poor prognosis (20,8) Recent studies on Ang-2-VEGF-A CrossMab showed The efficient and less harm effects on animals proposed that it characterized a new and active therapeutic chance for patients with malignancy with the probability to replace Bevacizumab as a

CONCLUSION
Ang-2 was overexpressed in OSCC and significantly associated with histopathological grade of the tumor.