Suha Final.doc
J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical
Oral Diagnosis 94
Immunohistochemical expression of P16 and HER2/neu in
normal oral mucosa, oral epithelial dysplasia, and oral
squamous cell carcinoma
Suha ali Ahmed, B.D.S. (1)
Ahlam Hameed Majeed, B.D.S., M.Sc. (2)
ABSTRACT
Background: Oncogenesis in the oral cavity is widely believed to result from cumulative genetic alterations that
cause a transformation of the mucosa from normal to dysplastic to invasive carcinoma. The p16 gene produces p16
protein, which in turn inhibits phosphorylation of retinoblastoma (Rb), p16 play a significant role in early
carcinogenesis. A number of epidermal growth factor receptor (EGFR) family, HER2/neu, has received much
attention because of its therapeutic implications. The aims of the study were to evaluate and compare the
immunohistochemical expression of the cell cycle protein P16 INK4a and c-erbB2 (HER2/neu) in NOM, OED, and
OSCC. Correlate both marker expression with each other as well as with various clinicopathological findings.
Materials and methods: Sixty two formalin-fixed, paraffin embedded tissue blocks (20 cases of normal oral mucosa,
17 cases of oral epithelial dysplasia, and 25 cases of oral squamous cell carcinoma) were included in this study, an
immunohistochemical staining was performed using anti p16 monoclonal antibody, and anti HER2/neu polyclonal
antibody.
Results: Positive IHC expression of p16 was found in 18 cases (90%) of NOM, 16 cases (94.1%) of OED and in 20 cases
(80%) of OSCC. Positive IHC expression of HER2/neu was almost undetectable in NOM, while it was found in 9 cases
(52.9%) of OED, and in 15 cases (60%) of OSCC.The correlation between the expression of both markers were
statistically highly significant in NOM, significant in OED, and non significant in OSCC.
Conclusions: This study signify the important role of p16 and HER2/neu in oral carcinogenesis and in the evolution of
the mucosa from normal to dysplastic to invasive carcinoma
Keywords: NOM, OED, OSCC, P16, HER2/neu. (J Bagh Coll Dentistry 2014; 26(2): 94-98).
الخالصھ
التي تحول الغشاء المخاطي الفموي و بخطوات عدیده من الطبیعي الى الحثل النموي عملیة التسرطن في التجویف الفمي غالبا ما تحدث نتیجة تراكم التغییرات الجینیھ : الخلفیھ
یلعب دورا مھما في عملیة p16وكذلك فان بروتین , Rbلمقابل یمنع فسفرة بروتین الذي في ا P16فھو ینتج بروتین p16 المورث الجیني.الطالئي ثم النسیج السرطاني المتغلغل
. والذي حظي اھتماما كبیرا بسبب تطبیقاتھ العالجیھ) EGFR( فھو أحد افراد عائلة مستقبالت عامل نمو خالیا األدمھ HER2/neu أما .التسرطن المبكر
و الحثل النموي الطالئي الفموي ,في النسیج المخاطي الفموي الطبیعي HER2/neuومحفز p16 INK4aسیجي الكیمیائي لبروتین تقییم التعبیر المناعي الن :اھداف الدراسھ
مع المقاییس السریریھ والمرضیھ مع بعضھما وكذلك عالقتھما HER2/neuو p16 وكذلك اكتشاف عالقة المورثات الجینیھ . وسرطان الفم الحرشفي ,
حالھ من الحثل النموي الطالئي الفموي 17حالھ من الغشاء الفموي الطبیعي و 20تضمنت , تشمل الدراسھ اثنان وستون حالھ مختاره من قوالب شمع البارافین:اد وطرق العمل المو
. HER2/neu و p16و اجراء الفحوصات المناعیھ الكیمیائیھ النسیجیھ باستخدام المؤشرات .حالھ من سرطان الفم الحرشفي 25و
من الغشاء الفموي الطبیعي و الذي اظھر تعبیر %)90(حالھ 18كان قد تبین في p16لبروتین اظھرت الدراسھ الحالیھ ان التعبیر االیجابي المناعي النسیجي الكیمیائي: لنتائج ا
من سرطان %) 80(حالھ 20في p16بینما كان التعبیر االیجابي لبروتین . ,لطالئي الفموي من الحثل النموي ا%) 94.1(حالھ 16كما وظھر التعبیر االیجابي في , ایجابي ضعیف
قد تبین HER2بینما في الحثل النموي الطالئي الفموي كان الظھور االیجابي ل محفز , لم یتم الكشف عنھھ في الغشاء الفموي الطبیعي HER2التعبیر االیجابي لمحفز .الفم الحرشفي
p16العالقھ االحصائیھ بین المؤشرین .من سرطان الفم الحرشفي%) 60 (حالھ 15كان قد وجد في HER2بینما التعبیر االیجابي لمحفز , %)52.9( حاالتفي تسع
حین انھ لم تكن ھناك عالقھ معنویھ بین المؤشرین في , وكذلك كانت العالقھ معنویھ في الحثل النموي الطالئي الفموي , كانت معنویھ للغایھ في الغشاء الفموي الطبیعي HER2/neuو
.في سرطان الفم الحرشفي
في عملیة التسرطن وكذلك في تطور الغشاء المخاطي الفموي من الطبیعي الى الحثل النموي HER2/neuومحفز p16كشفت ھذه الدراسھ على الدور المھم لبروتین :االستنتاجات
. ثم الى سرطان الفم الحرشفي
INTRODUCTION
Oral carcinogenesis is a multi-step process
involving gene mutations and chromosomal
abnormalities (1). The transition from normal oral
epithelium to oral dysplasia and cancer results
from accumulated genetic and epigenetic
alterations (2).
The best-known precursor lesion is epithelial
dysplasia, which is histologically detectable and
often presents clinically as white or red mucosal
patches called leukoplakia and erythroplakia (3).
(1) Master student, Department of Oral Diagnosis, College of
Dentistry, University of Baghdad.
(2) Professor, Department of Oral Diagnosis, College of
Dentistry, University of Baghdad.
Oral cancers account for nearly 3% of all
malignancies, and they are the sixth cancer by
incidence worldwide, with epidemiologic
variations existing between different geographic
regions (4-6).
The cell cycle protein (p16) is a well-known
tumor suppressor protein, composed of 156 amino
acids encoded by a three exons of the p16 gene. It
regulates the Rb tumor suppressor pathway by
keeping Rb in a hypophosphorylated state, which
further promotes the binding of E2F to achieve
G1 cell-cycle arrest. (7-9).
The transmembrane tyrosine kinase receptor
constitute the ErbB receptor family and comprised
of four di erent receptors known as ErbB1 (also
referred to as (EGFR), ErbB (HER2/neu in
rodents), ErbB3 (HER3), and ErbB4 (HER4) (10-
J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical
Oral Diagnosis 95
12). C-erbB-2proto-oncogene (HER/NEU/neu)
encodes a 185 transmembrane protein product of
tyrosine kinase family, with an extensive
homology to the epidermal growth factor receptor
(13) and can be activated by hetero oligomerization
with the other members of the ErbB family (14).
The lack of a unique marker of OSCC has long
been a problem in the early detection of OSCC. It
would be necessary to discover more reliable and
efficient markers to characterize the malignant
transformation of oral epithelium (15, 16).
This study aimed to evaluate and compare the
expression of P16 and HER2/neu in normal oral
mucosa, oral epithelial dysplasia, and oral
squamous cell carcinoma, and to correlate both
marker expression with each other, as well as with
various clinicopathological findings including
(age, sex, clinical presentation, tumor site, tumor
grade).
MATERIALS AND METHODS
The study samples included sixty two
formalin-fixed, paraffin embedded tissue blocks
(20 NOM, 17 OED, and 25 OSCC) dated from
(1975 till 2013), were obtained from the archives
of the department of Oral & Maxillofacial
Pathology/ College of Dentistry/ University of
Baghdad; Al-Shaheed Ghazi Hospital/ Medical
City / Baghdad; and Al Kadhimiya teaching
Hospital. Sections of 4µm thickness were
mounted on normal glass slides, stained with
H&E and histopathologically re-evaluated. Four
other 4µm thick sections for each case were cut
and mounted on positively charged slides (Fisher
scientific and Escho super frost plus, USA) for
immunohistochemical staining with monoclonal
antibody p16 using Abcam expose mouse and
rabbit HRP/DAB immunohistochemical detection
kit (Catalog No. ab54210, Cambridge, UK). And
polyclonal antibody HER2/neu using Rabbit Anti-
Human c-erbB-2 Oncoprotein (Catalog No. A
0485) Dako Denmark immunohistochemical
detection kit was used.
RESULTS
Positive p16 Immunostaining was detected as
brown nuclear or (nuclear and cytoplasmic)
expression.
IHC staining of p16 in NOM reveals that 2
cases (10%) showed negative expression, 18
(90%) cases showed weak positive expression.
And in OED, 1 case (5.9%) showed negative
expression, 1 case (5.9%) showed weak positive
expression, 6 cases (35.3%) showed moderate
positive expression, and 9 cases (52.9%) showed
high positive expression. While in OSCC, IHC
staining of p16 reveals that 5 cases (20%)
showed negative expression, 3 cases (12%)
showed weak positive expression, 2 cases (8%)
showed moderate positive expression, and 15
cases (60%) showed high positive expression. fig
(1,2,3)
Positive HER2/neu immunostaining was
detected as brown membranous or (membranous
and cytoplasmic) expression. Regarding
HER2/neu expression in NOM, all cases (100%)
showed negative expression. And in OED, 8 cases
(47.1%) showed negative expression, 5 cases
(29.4%) showed weak positive expression, and 4
cases (23.5%) showed strong positive expression.
While in OSCC HER2/neu immunostaining
reveals that 10 cases (40%) showed negative
expression, 10 cases (40%) showed weak positive
expression, and 5 cases (20%) showed strong
positive expression. Fig (4,5,6).
Regarding the correlation between p16 and
HER2/neu expression in each group and
according to Mann-Whitney U test, the results
revealed a statistically highly significant
correlation in NOM (p-value= 0.000), and
significant correlation in OED (p=0.02), while the
results revealed non significant correlation in
OSCC (p=0.14). As clarified in table (1).
Regarding groups’ comparison in each marker,
the results revealed statistically highly significant
correlation (p=0.000). As clarified in table (2).
Figure 1: Positive nuclear expression of p16
in NOM (40x).
Figure 2: Positive nuclear & cytoplasmic
expression of p16 in severe dysplasia (40x).
J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical
Oral Diagnosis 96
Figure 3: positive nuclear expression of p16
in moderately differentiated SCC,(40x).
Figure 4: Negative HER2/neu expression in
NOM (tongue) (40x)
Figure 5: Positive membranous &
cytoplasmic expression of HER2/neu in
severe dysplsia (10x).
Figure 6: Positive membranous &
cytoplasmic expression of HER2/neu in
moderately differentiated SCC (10x)
Table 1: Descriptive statistics and markers’ comparison in each group
Groups Markers Descriptive Statistics Comparison N Mean S.D. S.E. Mann-Whitney U test p-value
NOM P16
20 12.05 5.86 1.31
-3.93 0.000 (HS) HER2 20 4.65 2.98 0.67
ED P16
17 62.24 32.98 8.00
-2.36 0.02 (S) HER2 17 32.24 30.93 7.50
SCC P16
25 52.20 36.16 7.23
-1.47 0.14 (NS) HER2 25 31.88 29.71 5.94
Table 2: Descriptive statistics and groups’ comparison in each marker
Markers Groups
Descriptive Statistics Comparison
N Mean S.D. S.E. Kruskal-Wallis test p-value
P16
NOM 20 12.05 5.86 1.31
20.66 0.000 (HS) ED 17 62.24 32.98 8.00
SCC 25 52.20 36.16 7.23
HER2/neu
NOM 20 4.65 2.98 0.67
18.29 0.000 (HS) ED 17 32.24 30.93 7.50
SCC 25 31.88 29.71 5.94
DISCUSSION
This study is not a large epidemiological one
that expressed the incidence and prevalence of
different clinicopathological features of OED and
OSCC, however, there was a close correlation
between the present data and other published data
concerning the incidence of OED and OSCC in
J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical
Oral Diagnosis 97
Iraq in the past studies records and studies in other
parts in the world (17,18).
Assessment of p16 immunohistochemistry
The results of the present study showed that
positive immunostaining of p16 was found in
90% of normal oral mucosa cases with variable
nuclear and cytoplasmic expression. This result
agrees with Tarakji et al. (19).
As p16 is involved in cell cycle regulation its
expression may vary with cell turnover times in
the oral mucosa which have been shown to be
variable in different types of oral mucosa (20). The
activation of p16 expression can be triggered by
DNA damage, oncogenic stress or physiological
aging (21).
Concerning OED cases the results of this study
showed that positive expression of p16 was
observed in (94.1%) of OED cases. Regarding
correlation between p16 and clinicopathological
features, there was statistically non significant
correlation, which agreed with Bradley et al. (22).
P16 positivity was found in (80%) of OSCC
cases. Concerning the correlation between
clinicopathological findings of OSCC cases and
p16, the present study showed statistically
significant correlation between p16 expression
and the tumor site. While there was non
significant difference between p16 with age, sex,
site, and grades of OSCC (23,24). These differences
due to limited sample size of this study.
Different cancer-causing agents may lead to
p16INK4a gene inactivation as well as altered p53
and pRb tumor suppressive pathways (25,26). These
changes may result in either loss or
overexpression of p16INK4a in oral dysplasia and
OSCC.
HPV oncogenes are frequently found in
oropharyngeal squamous cell carcinomas that
display concomitant increased p16 INK4a
expression (27,28). Other relevant etiopathological
agents that may influence p16 INK4a expression are
smoking and smoke-less tobacco use (19,27). The
oral mucosa of smokers, express p16 INK4a more
frequently when compared to individuals that do
not use tobacco, and this could be attributed to the
component of tobacco smoke (nicotine), which is
well known to significantly stimulate cell growth,
epithelial cell DNA synthesis and cell
proliferation that stimulated at nicotine
concentrations lower than those obtained in blood
after smoking (27,29).
Assessment of HER2 / neuimmuno-
histochemistry
The present study showed negative HER2/neu
immunoreactivty in normal oral mucosa. And in
OED it was found in (52.9%). These results were
in agreement with Jubair (18) that showed
HER2/neu expression in NOM was almost
undetectable. According to clinicopathological
correlation of HER2/neu and OED, the results of
this study showed statistically non-significant
correlation, which agrees with Jubair (18).
HER2/neu positivity was found in (60%) of
OSCC cases. Agree with (18) that showed higher
HER2/neu expression in the OSCC group.
The overexpression of HER2/neu could be a
potential useful marker in distinguishing non-
cancer and cancer, as shown in this study. Once
the overexpression of HER2/neu is found in cases
with benign or precancerous lesions in the oral
cavity, care should be taken in the follow-up of
such patients. Early treatment with excision of the
ED showing expression of HER2/neu may be
required (30,31). Activation of EGFR family by a
variety of ligands is necessary for normal growth
and differentiation (32).
The present study showed statistically highly
significant correlation between p16 and
HER2/neu in NOM, and significant correlation
between them in OED, and showed statistically
non significant correlation between them in
OSCC.
Correlation between p16 and HER2/neu in
each group
This is the first study in Iraq and other parts in
the world assessing the correlation between p16
and HER2/neu immunohistochemical expression
in NOM, OED, and OSCC. Since this is a
pioneer research in assessing that correlation, so
the comparison could be withdrawn from other
studies using another technique ,which is agree
with (33) that showed there was non-significant
correlation between p16 deletion and HER2/neu
amplification in oral squamous cell carcinoma by
using fluorescent in situ hybridization.
The present study showed highly significant
correlation in each marker regarding groups’
comparison. This means that p16 and HER2/neu
play a role in oral carcinogenesis.
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