J Bagh College Dentistry Vol. 31(4), December 2019 Angiopoietin-2

Angiopoietin-2 Immunohistochemical Expression in Oral

Squamous cell Carcinoma

Intisar Abdel-Jabbar Al-Sarraf, B.D.S.(1)

Ban F. Al-Drobie B.D.S., M.Sc., Ph.D. (2)

ABSTRACT
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 formalin- fixed, paraffin- embedded 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.

Key words: OSCC, angiogenesis, Ang-2. (Received: 15/1/2018; Accepted: 19/2/2018)

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)

1. Master’s Student, Department of Oral Diagnosis, College of

Dentistry, University of Baghdad.

2. Assistant Professor, Department of Oral Diagnosis, College
of Dentistry, University of Baghdad.

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.

MATERIAL AND METHOD
Tissue Sample
Thirty six retrospective formalin- fixed, paraffin-

embedded tissue blocks diagnosed

histopathologically as OSCC were enrolled in this

study. The blocks were obtained from the archives

of the Oral Maxillofacial Pathology Department /

Dentistry Collage of Baghdad University.

Demographic and clinical data : patients name,

age, gender, clinical presentation, , and tumor site

were obtained from the archive. Normal placental

tissue of human for Ang-2 antibody

immunohistochemical detection obtained from

Al-Shaheed Ghazi Hospital, Teaching

Laboratory Department/ Baghdad Medical City

J Bagh College Dentistry Vol. 31(4), December 2019 Angiopoietin-2

Figure 1: Immunohistochemical expression

ofAng-2 in normal human placental

tissues(X100).

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

4o 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

37oC.

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(𝑥2).
Statistical significance was considered whenever

the P value was less than 0.05.

RESULT
This study included thirty six of

histopathologically confirmed OSCC cases, males

were 20 (55.6%) while female were 16 (44.4%).

Patients age ranged between 22-83 years, and the

mean age was 52.4 years. The most predominant

age group was (70-79) years which account 10

cases (27.78%). Clinically the study sample was

presented most predominantly as an Ulcer (21

cases of 58.33%), while mass compromised 15

cases(41.67%). The most predominant affected

site with tumor was tongue of 17 cases (47%)

followed by floor of mouth account 7 cases

(19%). Most predominant histopathological grade

was moderately differentiated as 14 cases

(38.89%), followed by well differentiated as 13

cases (36.11%), lasted with poorly differentiated 9

cases (25%).

Table (1) showed the association between gender

and site, the association was found to be

statistically non-significance (P value>0.05).

Table1: Distribution of study sample

according to site and gender.

SITE

Gender

Male Female Total

N % n % n %

Tongue 9 45.00 8 50.00 17 47.22

Floor of

the mouth
2 10.00 5 31.25 7 19.44

Buccal

mucosa
2 10.00 2 12.50 4 11.11

Mandible 3 15.00 0 .00 3 8.33

Soft

palate
2 10.00 0 .00 2 5.56

Hard

palate
1 5.00 1 6.25 2 5.56

Alveolar

ridge
1 5.00 0 .00 1 2.78

Total 20 100.00 16 100.00 36 100.00

χ2= 6.986 d f=6 p= 0.322

Table (2) showed that moderately differentiated

OSCC was the most predominant
histopathological grade account 14 cases.

J Bagh College Dentistry Vol. 31(4), December 2019 Angiopoietin-2

Table 2: The distribution of study sample

according to the histopathological grade of

the Tumor.
Grade n %

well differentiated 13 36.11

Moderate differentiated 14 38.89

Poor differentiated 9 25.00

Total 36 100.00

Table (3) showed the distribution of study sample

according to Ang-2. Half of cases with moderate

staining, those with faint and strong staining

represented (8,9 cases respectively), and one case

only with no staining.

Table 3: SIS of Ang-2 antibody

immunohistochemical expression in the study

sample

% n Score

SIS

(Ang-2)

2.78 1 0

22.22 8 1

50 18 2

25 9 3

100 36 Total

Ang-2: Angiopoietin-2, SIS: staining intensity

score. 0: No staining.1: faint staining.2: Moderate

staining. 3: Strong staining.

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 4: Association between Ang-2

expression and Age group in the study

sample

Age

groups

Ang-2

staining
Faint Moderate Strong Total

N % N % N % N % N %

<0r=39 0 .0 1 12.5 6 33.3 0 .00 7 19.4

40-49 0 .0 1 12.5 3 16.7 1 11.12 5 13.9

50-59 1 100 2 25. 0 .00 2 22.22 5 13.9

60-69 0 .0 2 25. 4 22.2 3 33.33 9 25.0

>0r=70 0 .0 2 25. 5 27.8 3 33.33 10 27.8

Total 1 100 8 100 18 100 9 100 36 100

𝑥2=14.184 d f =12 P=0.289

Table 5: Association between Ang-2

expression and Gender of the study group

Gender

Ang-2

staining
Faint Moderate Strong Total

N % N % N % N % N %

Male 1 100. 5 62.5 11 61.11 3 33.33 20 55.56

Female 0 .00 3 37.5 7 38.89 6 66.67 16 44.44

Total 1 100. 8 100. 18 100. 9 100. 36 100.

𝑥2= 2.981 df-3 P=0.395

Table 6: The association between Ang-2

expression and Clinical presentation of

OSCC.

Clinical

Presentation

Ang-2

staining
Faint Moderate Strong Total

N % N % N % N % N %

Ulcer 1 100. 6 75. 8 44.4 6 66.7 21 58.3

Mass 0 .00 2 25. 10 55.6 3 33.3 15 41.7

Total 1 100. 8 100. 18 100. 9 100. 36 100.

𝑥2 =3.314 d f=3 P=0.346

Table 7: Association of Ang-2 expression and

Site of distribution of study sample.

SITE

Ang-2

staining
Faint

Moderat

e
Strong Total

N % N % N % N % N %

Tongue 0 .00 5 62.5 8 44.4 4 44.5 17 47.2

Floor of

mouth
0 .00 0 .00 6 33.2 1 11.1 7 19.4

Buccal

mucosa
1 100 0 .00 1 5.6 2 22.2 4 11.1

mandible 0 .00 2 25. 0 .00 1 11.1 3 8.3

Soft palate 0 .00 1 12.5 1 5.6 0 .00 2 5.6

hard

palate
0 .00 0 .00 1 5.6 1 11.1 2 5.6

Alveolar

ridge
0 .00 0 .00 1 5.6 0 .00 1 2.8

Total 1 100 8 100 18 100 9 100. 36 100

𝑥2=21.852 d f=18 P=0.239

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).

J Bagh College Dentistry Vol. 31(4), December 2019 Angiopoietin-2

Table 8: The association between Ang-2

expression and the grade of the study

sample.

X2=14.674 d f=6 P=0.023* 𝑥2=
Ang-2: Angiopoietin-2. W.D: Well Differentiated.

M.D: Moderately Differentiated. P.D: Poorly

Differentiated. *: P Value<0.05.

Figure 1: No staining (Score0 in SIS ) of

Ang-2 expression of the studied samples.

(X100).

Figure 2: Faint staining (score1 in SIS) of

Ang-2 expression in the studied samples

(X100).

Figure3: Moderate staining (score2 at SIS) of

Ang-2 expression in studied samples (X100).

Figure4: Strong staining(Score3 in SIS) of

Ang-2 expression of the studied samples

(X100).

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 "(16, 17, 18)

Grade

Ang-2

No Stain Faint Moderate Strong Total

N % N % N % N % N %

W.D 0 .00 7 87.5 4 22.2 2 22.2 13 36.1

M.D 0 .00 1 12.5 9 50. 4 44.4 14 38.9

P.D 1 100 0 .00 5 27.8 3 33.3 9 25.

Total 1 100 8 100. 18 100. 9 100. 36 100.

J Bagh College Dentistry Vol. 31(4), December 2019 Angiopoietin-2

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.

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J Bagh College Dentistry Vol. 31(4), December 2019 Angiopoietin-2

الخالصة
و تقىىم عالقته بالنواحى السرىرىة والمرضية لسرطان الفم الحرشفى Angiopoietin-2الظهور الكىمىائى النسىجى المناعى لمعلم

ويعتقد Angiopoietin-2(Ang-2)الخلفىة:هناك الكثىر من البرويىنات التي تفرز والتي لها تاثيرفي توقع نتائج مرض سرطان الفم الحرشفي واحد هؤالء هو المعلم

ان له دور اساسي في نشوء وتفاقم هزا النوع من السرطان.

في سرطان الفم الحرشفي وتقييم ارتباطه بالنواحي السريرية والمرضية والتمايز Ang-2اهداف الدراسة: ان هذه الدراسة تشير الى تحديد الظهور المناعي لمعلم

النسيجي لهدا النوع من الورم.

الدراسة ست وثالثون عينة مسترجعة استخرجت من المقاطع النسيجية المطمورة بشمع البرافين ثم اجريت عليها الفحوصات النسيجية الطرق و االدوات:تضمنت

وقرات النتائج باستخدام طريقة حساب شدة التصبيغ شبه الكمية. Ang-2 والمناعية النسيجية الكيميائية لمعلم

( (P value=0.023وارتباطه مع الدرجة النسيجية المرضية والعالقة ذو داللة معنوية Ang-2يالنتائج: بينت النتائج ظهور المعلم المناع

معنوية مع النواحي السريرية والمرضية لسرطان الفم الحرشفي. كما اوضحت هذه الدراسة عدم وجود داللة

في تقييم السلوك البيولوجي وتخميين نتائج المرض. ا يشير الهميتهوهذ Ang-2االستنتاجات: كشفت هده الدراسة مستويات مرتفعة من التعبير المناعي لمعلم