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114 Medico-legal Update, October-December 2020, Vol. 20, No. 4
The Meshing between Epstein Barr Virus Nuclear
Antigen-1 and P53 in Iraqi Malignant Breast Tissues
Bareq N. Al-Nuaimi
Assistant Lecturer (M.Sc. in Microbiology), Department of Biology,
Madenat Alelem University College, Baghdad, Iraq
Abstract
In Iraq, breast cancer incidence exceeds any other type of cancers and the etiology not understood well.
Epstein Barr virus is a gamma herpesviruses and one of carcinogenic viruses that may implicated to
breast carcinogenesis. The nuclear antigen-1 (EBNA-1) protein is the sole EBV antigen that presented
in all tumors related to EBV and plays pivotal roles in carcinogenesis of the virus. Examination applied
by immunohistochemistry (IHC) to detect and demonstrate the correlation between (EBNA-1) and tumor
suppressor protein (P53) expression. The study includes paran-embedded tissue blocks of ninety 90
malignant breast tissues and thirty 30 normal breast autopsies. EBNA-1 was signicantly expressed in 40/90
(44.4%) of malignant tissues while its expression in normal breast tissues was negative in all tested cases.
The tumor suppressor protein P53 was showed negative expression in all normal breast tissues and positive
in 27/90 (30%) in malignant breast tissues. A signicant negative relationship (r=-0.420; P<0.05) revealed
between EBNA-1and P53 expression. These nding reveal that EBNA-1 was evident in malignant breast
tissues and demonstrate the interplay between EBV and p53 raising the possibility that viral infection may
be involved in carcinogenesis process.
Keywords: EBV, EBNA-1, P53, and Immunohistochemistry.
Introduction
Malignancy of the breast is the top distributed cancer
among females in Iraq with an estimation of 4,720
cases in 2015. The incidence of breast cancer generally
exceeds any other type of cancers in Iraq (33.5% of all
cancers in Iraq)[1]. The causality of breast cancer is not
yet clearly understood, but its incidence related to some
environmental factors like viruses such as EBV [2]. EBV
belongs to the gamma herpesvirus family, aects over
90% of the world’s population and one of the main viruses
to be directly involved in cancer genesis[2]. EBV has
been involved as a causative agent of many cancers like
Hodgkin’s disease, non-Hodgkin’s lymphoma, Burkitt’s
lymphoma,, as well as nasopharyngeal carcinoma,
arising in immunocompromised individuals[3].
Researches revealed a strong relationship between EBV
and breast carcinoma by showing strong evidences[4].
In both primary and metastatic tumors, the constant
expression of some of EBV proteins suggests that
these proteins are the key player in the EBV-associated
tumorigenesis[5]. The tumor suppressor gene, p53 is
frequently related to many human cancers because most
of the human cancers have mutated p53 [6]. Mutations
in p53 happened in breast cancer and are typically
associated with more aggressive tumor characteristics,
but the clinicopathological and epidemiological features
of p53 protein expression is still not clear yet. Some
studies suggested that EBV infection induces p53
expression in away or another without causing mutations
[7]. Transformed cells by EBV are delicate to apoptosis
by P53 mediated pathways[7]. EBV infection of primary
cells usually induces a DNA damage-signaling pathway
and inhibits cellular proliferation[8]. At the same time,
EBV has the capacity to cancel the eect of p53 by
multiple means. EBV has many genes that blocks p53-
mediated apoptosis or downregulates the expression
of p53[9,10]. Epstein-Barr nuclear antigen-1 (EBNA1),
demonstrated in all tumors correlated with EBV, may act
as the sole protein in many tumors and supposed to be
implicated in tumorgenesis[20]. The present study used
Medico-legal Update, October-December 2020, Vol. 20, No. 4 115
to investigate the relationship between EBV-infected
patients by testing (EBNA-1) antigen expression with
p53 expression in Iraqi breast cancer patients.
Materials and Method
Patients and sampling: The present study have
done in a period between January 2019 and October
2019 in Baghdad. Paran blocks of breast biopsies
were obtained from patients of breast carcinoma after
mastectomy. Blocks were collected by a pathologist
from Al Alweiya teaching hospital according to ethical
considerations and the hospital approval. Formalin xed,
paran embedded tissue blocks of (90) malignant breast
tissue and (30) normal breast autopsies were obtained. A
faculty pathologist re-examined all slides stained with
Hematoxylin and Eosin (H&E) for histopathological
diagnosis of the chosen blocks.
Immunohistochemistry (IHC): Paran-
embedded blocks sectioned using a microtome to 4
μm and placed in tissue oatation bath (40C°) and then
placed on positively charged slides. The slides were set
in oven at 70C° for one hour followed by a series of
sequential xylene/ethanol/water washes that remove the
wax and rehydrate the tissue for subsequent antibody
binding. Heat-induced epitope retrieval (HIER) was
applied for antigen unmasking. Heating to boiling in
buer was applied for the slides using a pressure cooker
for 30 minutes. Then, the slides were transferred to PBS
(phosphate buer saline) before immunostaining. The
sections quenched in a hydrogen peroxide (H2O2) by
adding drops on the slides for 10 minutes. Protein block
solution were applied to each tissue section and allowed
it to remain in place for 10 minutes. Then, antibody
solution was added to the slides by using micropipette
100-200µl of diluted antibody solution. The slides were
incubated overnight (18-22) hour at room temperature.
Streptavidin peroxidase and biotinylated Goat Anti-
Mouse was added to each slide and incubated for
two hours each respectively. Washing of the slides
by washing buer should be applied after each stain.
Freshly prepared DAB chromogenic solution was added
to the tissue sections and incubated for 10 minutes. The
slides washed by distilled water and counterstained by
hematoxylin. Aqueous mounting media was added to
cover slips where the slides laid on. The slides left for a
while to be dried and be ready for examination under the
light microscope.
Results
Patients and sampling: A number of (90) malignant
breast tissues and (30) normal breast autopsies were
included in this study for sampling. Formalin-xed
paran embedded tissue blocks of breast obtained from
each woman included in this study and according to
ethical approval.
Epstein-Barr virus nuclear antigen expression
(EBNA-1): All malignant and normal tissue blocks were
tested for EBV nuclear antigen (EBNA-1) expression.
From 90 breast cancer samples, 40 cases (44.4%) were
positively express the antigen while all the normal control
cases were show negative expression (gure1and 2).
Our results show that EBNA-1 expression is signicant
in malignant breast tissue as compared to normal control
specimens (P<0.05).
Figure 1: 40X strong EBNA-1 antigen expression
of nuclear and cytoplasmic staining in breast
carcinoma, brown colour is the positive result.
Figure 2: 10X negative EBNA-1 antigen expression
in breast carcinoma. Brown colour is not present.
116 Medico-legal Update, October-December 2020, Vol. 20, No. 4
P53 Expression: P53 have been tested in all
cases by immunohistochemistry (IHC). From (90)
cases of breast carcinoma, a number of 27 (30%)
cases were express P53 positively. Among them, 17
(18.8%) showed strong expression, 10 (11.1%) cases
moderately express p53 (gures3, 4, and 5). According
to ages, The protein expressed in 18.8% (n=17) females
of age younger than or equal 50 years (≤ 50 years),
While females of ages older than 50 years the results
of expression 11.1% (n=10). A statistical signicant
(P<0.05) relation between patients ages and expression
of p53. In addition, It was found 14.4% (n=13) of p53
positive expression were of the histological grade III,
12.2% (n=11) of histological grades II and 3.3% (n=3)
of histological grade I. Statistical analysis was found to
be statistically signicant (P<0.05).
Mentioning lymph node involvement, 18.8% (n=17)
of positive cases were correlated with lymph node
involvement. P53 positive cases with no lymph node
involvement recorded in 11.1% (n=10). No signicant
association have been observed (p>0.05). The tumor size
also determined in the present study, It was found that
the size range > 10cm was the highest (15.5%, n= 14)
compared to other tumor size ranges of p53 expressed
cases. Statistics of estrogen and progesterone receptors
found in this study. From total number of malignant cases
in the study, A percentage of (20%, n=18) that express
p53 positively express estrogen negatively and only (10
%, n=9) express estrogen positively. The progesterone
receptor negative cases show higher p53 expression
(17.7%, n=16), whereas the positive cases seen only in
(n=11). Statistical association of p53 expressed cases
with hormone receptors, lymph nodes involvements and
tumor size were statistically not signicant (Table 1).
Table 1: Some clinic pathological factors and its relation to p53 expression in breast cancer patients
Parameter P53 expression
Positive (no. of patients) Negative (no. of patients) P-value
Lymph node metastasis
Node +
Node -
17
10
40
23
N.S
(P ˃ 0.05)
Tumor size
≤ 10 cm
> 10 cm
13
14
35
28
N.S
(P ˃ 0.05)
Estrogen receptor
Negative
Positive
18
9
42
21
N.S
(P ˃ 0.05)
Progesterone receptor
Negative
Positive
26
11
34
17
N.S
(P ˃ 0.05)
Figure 3: 40X Strong positive
Cytoplasmic and nuclear expression
of p53 in breast carcinoma. Dark
color showing the positive result
Figure 4: 40X moderate positive
Cytoplasmic and or nuclear staining
of P53 in breast carcinoma. Dark
color showing the positive result
Figure 5: 10X negative
p53 expression in breast
carcinoma, no dark brown
color appeared
Medico-legal Update, October-December 2020, Vol. 20, No. 4 117
The Correlation between EBNA-1 and P53
expression: Signicantly negative relationship (r=-
0.420; P<0.05) was recorded between (EBNA-1) antigen
expression and P53 protein expression in malignant
breast tissues. No other relationships noticed between
EBNA-1 antigen and the clinicopathological parameters
of malignant breast tissues.
Discussion
The present study revealed that EBV signicantly
expressed in malignant breast tissues than in normal
autopsies. EBNA-1 antigen demonstrated in 44.4% of
the breast cancer cases, which indicated that EBV might
have a role in breast carcinogenesis. A previous studies
support the present indication, serological researches on
breast cancer patients on EBV relation to disease using
the traditional marker of EBV revealed that EBV IgG
levels score (97/208; 96%)[11]. Another study tested for
both anti-EBNA-1 IgG level by ELISA technique and
EBNA-1 antigen expression by immunohistochemistry
technique (IHC), the results show that 90.9% of breast
cancer cases were seropositive for anti-EBNA-1 IgG
and EBNA-1 was positively expressed in 28/51 cases
(54.9%) by IHC[12]. The nuclear antigen 1 (EBNA1)
protein is the only EBV protein that presented in all
tumors that are associated with EBV and plays many
important roles in the latency of the virus. The cellular
processes that the viral protein play is to reduce apoptosis
and increase cell survival [15].
Determining the P53 expression is important
for aiding in diagnosis and helping to decide the best
therapeutic way in addition to predicting prognosis[13].
Although our present study found signicant
association of p53 expression with tumor grade, it
demonstrated non-signicant relationships with other
tumor parameters. Previous reports indicated that, P53
expression correlated with clinical grading of the disease
and age of the patients signicantly. No signicant
statistical correlations observed with lymph node
involvement, tumor size, and expression of estrogen and
progesterone receptors[21,14]. In spite, these associations
were not signicant in statistical manners of the present
study, the contrasts in the results explained to genetic,
environmental, and possibly social factors [12,13].
All the previous reports of p53 expression in
malignant breast tissues extended approximately from
10% to 70% [12]. During this project, p53 demonstrated
in 30% of the samples. These investigations can be
compared to previous researches exclusively regarding
the associations with histological grading and hormonal
status. The relatively high expression of p53 could be
related to genetic and environmental parameters that
reveals the mutation type of P53. In addition, Most of the
cases at relatively advanced stages of the disease when
they diagnosed for the rst time and are already at higher
expression of P53. That may reect the bad prognosis
in many of the present study cases [11]. P53 is a tumor
suppressor gene, mutations in gene commonly occur in
breast cancer. Alterations in the gene lead to change the
expression of many other genes that controlled directly
or indirectly by p53. The results are malfunctioning of
DNA damage repair pathways, cell-cycle arrest, and
apoptosis[16].
The relationship between EBNA-1 and P53 is
not clear yet. A study in Brazil found that there was a
correlation between EBNA-1 and p63 expression, but
not between EBNA-1 and p53 [17]. Our present study
revealed that there is a signicant negative relationship
between (EBNA-1) antigen expression and P53 in breast
cancer patients meaning that increased expression of
EBNA-1 lowers p53 expression. A study, dealt with a
key regulator of p53 (USP7), determined that EBNA1
binding to USP7 lowers p53 levels and protect cells from
apoptotic challenge [18]. A researchers in (2019) found
that EBNA1 lowers p53 level in osteosarcoma cell [19].
Conclusion
The present study revealed Epstein Barr virus nuclear
antigen-1was signicantly expressed in malignant
breast tissues and this raises the possibility that EBV
may have a role in carcinogenesis of the breast. Tumor
suppressor protein P53 expressed in about 30% of the
malignant cases. The correlation between EBNA-1 and
P53 expression was negatively signicant which means
that increasing the expression of EBNA-1 in malignant
breast tissues may lower the tumor suppressor protein
P53. This foundation may explain one the EBV roles in
tumorgenesis of the breast in Iraqi patients.
Conict of Interest: None
Funding: Self
Ethical Clearance: Not required
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