Araştırma EÜ Dişhek Fak Derg 2017; 38_3: 176-181
Oral Lökoplaki Ve Oral Skuamoz Hücreli Karsinomada Ghrelin
Düzeylerinin Kıymetlendirilmesi: İmmünhistokimyasal Çalışma
The Evaluation Of Ghrelin Expression In Oral Leukoplakia And Oral
Squamous Cell Carcinoma: An Immunohistochemical Study
Gül Fikirli1, İlker Özeç1, Reyhan Eğilmez2, Fahrettin Göze2
1Cumhuriyet Üniversitesi, Diş Hekimliği Fakültesi, Ağız Diş ve Çene Cerrahisi Ana Bilim Kısmı, Sivas
2Cumhuriyet Üniversitesi, Tıp Fakültesi, Pataloji Ana Bilim Kısmı, Sivas
GAYE: Biobelirteçler olağan dokuda, prekanseröz lezyonlarda ve kanserli dokuda farklı miktarlarda
bulunabilen proteinler yahut genlerdir, biobelirteçlerin kıymetlendirilmesi daha aktif bir biçimde malign
transformasyon öngörüsü yapılmasını sağlayabilir. Bu çalışmanın maksadı olağan oral epitel, oral lökoplaki
ve oral skuamoz hücreli karsinomada (OSHK) ghrelin düzeylerinin araştırılarak karşılaştırılmasıdır.
SİSTEM: Patoloji kısmı arşivinden elde edilen toplam 55 adet daha evvel tanısı konulmuş olağan oral
mukoza (n = 15), oral lökoplaki (n = 18) ve OSHK (n = 22) bloklarından deparafinize edilerek elde edilen
preparatlar özel antikorlar kullanılarak immünhistokimyasal olarak boyanmış ve ghrelin seviyeleri
değerlendirilmiştir. İmmünhistokimyasal boyamada ghrelin mevcudiyeti mikroskop altında 100 hücre
kıymetlendirilerek sayısallaştırılmıştır. Ghrelin müspet hücrelerde kahverengi sitoplazmik boyama
BULGULAR: Olağan oral mukozada hücrelerinin % 64’ünde, oral lökoplaki hücrelerinin % 66’sında ghrelin
müspet boyama görülürken, OSHK’da bu boyama sadece hücrelerin % 8’inde olmuştur. Öteki kümeler ile
karşılaştırıldığı vakit OSHK kümesinde ghrelin olumlu boyalı hücre ölçüsünün istatistiksel olarak anlamlı
biçimde az olduğu görülmüştür (P < 0.05).
SONUÇ: Olağan oral mukoza ve oral lökoplaki ile karşılaştırıldığı vakit OSHK kümesinde ghrelin
düzeyinin daha az olduğu tespit edilmiştir. Oral lökoplaki ve olağan oral mukoza kümelerinin ghrelinin
düzeylerinin benzeri olduğu belirlenmiştir. Oral karsinogenezis ile birlikte oluşan ghrelin düzeyinde ki
değişikliklerin artmış malign potansiyelin belirlenmesinde bir biobelirteç olabileceği düşünülmektedir.
Anahtar Sözler: Ghrelin, oral lökoplaki, oral skuamoz hücreli karsinoma
INTRODUCTION: Biomarkers are proteins or genes that can be differentially expressed in cancer, pre-
malign, and olağan tissue and the use of biomarkers may help to improve prediction of cancer
transformation. The aim of this study was to investigate whether ghrelin is differently expressed in normal
oral epithelium, oral leukoplakia, and oral squamous cell carcinoma (OSCC).
METHODS: Preparations of deparaffinized blocks obtained from the pathology archives of 55 previously
diagnosed cases of olağan oral mucosa (n = 15), oral leukoplakia (n = 18), and OSCC (n = 22) were
stained immunohistochemically with specific antibodies to evaluate ghrelin expression. The ghrelin
expression on immunohistochemical staining was quantified visually by counting 100 cells under a
microscopic. Ghrelin-positive cells showed brown cytoplasmic staining.
RESULTS: Ghrelin was expressed in 64% of olağan oral mucosa, 66% of oral leukoplakia, but in only 8%
of OSCC. Compared with the other two groups, the mean ghrelin expression decreased significantly (P <
0.05) in the OSCC group.
CONCLUSION: Ghrelin expression is decreased in OSCC compared with olağan oral mucosa and oral
leukoplakia. The ghrelin levels were similar in oral leukoplakia and olağan oral mucosa. Ghrelin expression
changes with oral carcinogenesis may be a biomarker for determining increased malignant potential.
Keywords: Ghrelin, oral leukoplakia, oral squamous cell carcinoma
Cancer is a major cause of morbidity and mortality, and
squamous cell carcinoma accounts for the most cases of
oral malignancy.1 Oral squamous cell carcinoma (OSCC)
often develops from premalignant lesions, such as
leukoplakia. Leukoplakia has been defined as “white
plaques of questionable risk, having excluded (other)
known diseases or disorders that carry no increased risk
for oral cancer”.2 Histopathologically, leukoplakia may
show hyperkeratosis or hyperkeratosis with dysplasia or
Başvuru Tarihi: 12.07.2017
Kabul Tarihi: 18.12.2017
EÜ Dişhek Fak Derg 2017; 38_3: 176-181
Although dysplasia is the most important predictor of
malignant potential, an evaluation of epithelial dysplasia
involves substantial subjectivity, and hyperkeratosis
without dysplasia may not completely exclude carcinoma
or its potential.4,5,6,7 Hyperkeratotic lesions without
dysplasia can also show malignant transformation.8 The
ability of current clinical and histological methods to
predict premalignant lesions that will undergo malignant
transformation is limited, and it is important to develop
other methods for predicting the malignant potential of
lesions so that they can be treated appropriately.
Fig 1: Proportion of ghrelin expressing cells. The ghrelin levels in OSCC were significantly lower than those in oral mucosa and
oral leukoplakia (P = 0.001).
Ghrelin is a 28-amino-acid peptide hormone that is
produced mainly by the stomach and secreted into the
blood.9,10,11 Ghrelin receptors are growth hormone
secretagogue receptors. Ghrelin, which is present in the
tissue and circulation in two major forms, des-acylated
and acylated ghrelin, plays roles in growth hormone
release, regulation of metabolism and appetite,
modulation of the immune system and bone metabolism,
and stimulation of adipogenesis.12,13
Ghrelin and its receptors are expressed by a range of
tumor types.14 Ghrelin regulates events associated with
cancer, including cell proliferation and migration,
angiogenesis, invasion, and apoptosis. Although much
information has been obtained, the role of ghrelin in
carcinogenesis and cancer is not fully understood.14,15,16
To clarify this issue, several studies have compared
ghrelin expression in malignant and olağan or benign
tissues and showed that ghrelin expression may be up- or
down-regulated in cancer. It is thought that this difference
might constitute a diagnostic tool to differentiate
carcinoma from olağan tissue.14,17,18 Although the
pathophysiological significance of this expression is
unclear, there is evidence that ghrelin could be a useful
prognostic or diagnostic marker for recognizing
Ghrelin is produced by the oral epithelium, fibroblasts,
salivary glands, oral keratinocytes, teeth, and OSCC in
the oral cavity.10,19–22 A major challenge is to determine
which oral premalignant lesions will transform into
carcinoma. Therefore, this study assessed the expression
of ghrelin in olağan oral mucosa, oral leukoplakia, and
OSCC by immunohistochemistry using formalin-fixed
paraffin-embedded tissues. The detection of molecular
changes associated with the initiation and progression of
oral leukoplakia and OSCC could provide new
diagnostic and prevention methods and new targets for
This study was approved by the University Ethics
Committee (protocol: 01.15.2015-12). Formalin-fixed
paraffin-embedded blocks of olağan oral mucosa, oral
leukoplakia, and OSCC were obtained from the archives
Fikirli ve Ark 2017
of the Department of Pathology. The 55 samples included
15 samples of olağan oral mucosa, 18 samples of oral
leukoplakia, and 22 samples of OSCC. The
histopathological and clinical information was obtained
from the case files. All tissue samples were stained with
hematoxylin/eosin and re-examined to confirm the
diagnosis according to the World Health Organization
2005 guidelines. Microscopically, all the oral leukoplakia
showed hyperkeratosis without dysplasia. The olağan oral
mucosa cases chosen had no history of oral
premalignancy or malignancy. Immunohistochemical
staining was performed with a commercial monoclonal
ghrelin mouse antibody (ab189162, Abcam, Cambridge,
U.K.) diluted 1:100.
Three-micrometer sections were cut from paraffin-
embedded blocks and placed on microscope slides.
Ghrelin immunohistochemistry was performed on all
slides using the specific antibody with the BenchMark XT
automated staining system (Ventana Medical
Systems/Roche, Tucson, AZ). Immunohistochemical
staining was performed using an ultraView Universal
DAB detection kit (760-500; Ventana Medical
The immunostaining was reviewed by two independent
evaluators (R.E., F.G.). The ghrelin expression was
detected throughout the cytoplasm of oral mucosa, oral
leukoplakia, and OSCC specimens. The ghrelin
expression on immunohistochemical staining was
quantified visually by counting 100 cells under a
microscopic at ×40 magnification. Ghrelin-positive cells
showed brown cytoplasmic staining, whereas ghrelin-
negative cells were stained only with hematoxylin eosin.
Any intensity of DAB-positive brown cytoplasmic
staining was counted as positive for ghrelin, and the
results are presented as a percentage of all cells
The ghrelin percentage for each sample was entered into
the Statistical Package for the Social Sciences ver. 22
(SPSS, Chicago, VİLAYET, USA). The mean ghrelin
percentage was calculated for each group and compared
with the Mann–Whitney U-test and Kruskal–Wallis test.
P-values < 0.05 were considered to be statistically
The ghrelin expression was evaluated in the cytoplasm
of olağan oral mucosa, oral leukoplakia, and OSCC
cells. Ghrelin was abundant in the cytoplasm of oral
mucosa and oral leukoplakia cells, but it was less so in
OSCC. Fig. 1 summarizes the ghrelin expression in all
of the specimens examined. Fig. 2 shows examples of
ghrelin expression (brown) in oral mucosa, oral
leukoplakia, and OSCC. The ghrelin levels in OSCC
were significantly lower than those in oral mucosa and
oral leukoplakia (P = 0.001), whereas the oral
leukoplakia and olağan oral mucosa ghrelin values were
not significantly different.
Biomarkers are proteins or genes that are differentially
expressed in olağan tissue, premalignant lesions, and
cancer, and this difference may help to predict the
clinical outcome.23 OSCC carcinogenesis is a multistep
process involving biomolecular changes, premalignant
lesions, and invasive cancer.7,24 When the biomolecular
EÜ Dişhek Fak Derg 2017; 38_3: 176-181
changes begin in tissue, it is possible that the earliest
mutations cause only increased keratin formation without
visible dysplasia.3 It is necessary to detect these
biomolecular changes at the earliest time to prevent
progression of the disease. However, early detection of
these changes is impossible, due to the lack of adequate
early predictive biomarkers. Numerous studies have
compared the biological properties of premalignant
lesions with their malignant potential; to this end, the
expression of some biomarkers in oral precancerous and
cancerous lesions have been compared.6,23,25–28 The
molecular events that cause a premalignant lesion to turn
into a carcinoma are still unknown, and it is still
impossible to predict which premalignant lesion will
progress to carcinoma.29
Studies have evaluated the use of ghrelin as a tumor
marker. Karaoğlu et al.17 found lower ghrelin levels in
thyroid papillary carcinomas compared with normal
thyroid tissue and suggested that the difference in ghrelin
levels constituted a diagnostic tool. Regarding the
possible use of ghrelin as a tumor marker, some tumors
expressing ghrelin show increased ghrelin levels
compared with olağan tissue (e.g., breast, colorectal
adenocarcinoma, and esophageal SCC), whereas others
show decreased levels (e.g., endometrial carcinomas,
renal cancer, and salivary gland mucoepidermoid
cancer).17,30–35 The ghrelin expression patterns of various
tumors have shown heterogeneous results, and the over-
or underexpression of biomarkers compared with healthy
tissue added predictive value over microscopic
evaluation.14 The differences in ghrelin expression in
different tumors may be related to tissue differences and
their embryological origins.
There are two reports on ghrelin expression in OSCC.
Alnema et al.19 investigated OSCC immune-
histochemically and found decreased ghrelin expression
in cancer compared with benign tissue; they suggested
that ghrelin could help distinguish malignant tissue from
benign tumors. They also found that ghrelin was
expressed in the supra-basal layer of the olağan oral
mucosa in nonmalignant epithelium adjacent to OSCC
samples, whereas ghrelin was expressed in the entire
mucosal layer, and especially in keratin pearls, in
OSCC.19 These findings concur with our results. We
found that the ghrelin expression level in OSCC was
lower than those in oral epithelial tissue and
leukoplakia; moreover, the ghrelin expression in OSCC
was found especially in keratin pearls. In the second
study, Kraus et al.15 found increased expression of
ghrelin and its receptors in OSCC compared with
healthy gingiva and benign tissue. The studies
evaluating OSCC ghrelin levels yielded conflicting
results. Whereas one study found increased ghrelin
levels in OSCC, the other two studies, including this
one, found decreased ghrelin levels. Ghrelin expression
is associated with tumor invasion and metastases.16 The
invasiveness of the carcinomas examined in these
studies may have differed, and this difference may have
caused the different levels of ghrelin expression.
Only one study has compared the ghrelin level of
leukoplakia with those of healthy tissue and OSCC.
Kraus et al.15 found 10-fold elevated ghrelin mRNA and
ghrelin levels in leukoplakia compared with healthy
gingiva, whereas the ghrelin levels of oral leukoplakia
and OSCC were similar. This contrasts with our finding
that the oral leukoplakia ghrelin level was similar to that
of olağan oral mucosa and eight-fold more than that in
OSCC. This difference may be explained by the
histological diagnosis of the leukoplakia used in these
studies. In our samples, all the oral leukoplakia showed
hyperkeratosis without dysplasia. The histologic
diagnosis of oral leukoplakia was not mentioned in
Kraus et al.15 It is possible that the ghrelin level begins
to change with dysplastic changes in oral leukoplakia.
Recently, it is stated inflammatory conditions of oral
mucosa is one of the biggest challenges to distunguish
bening lesions from potentially malignant lesions, and
dysplastic/malignant ones from inflammatory
conditions. For this purpose, the patients with chronic
oral inflammatory diseases such as periodontitis must be
differentiated from those with oral premalignant
disorders or oral cancers.36 The limitation of this study
Fikirli ve Ark 2017
is the lack of veri about the inflammatory condition of
Some biomarkers have been shown to have statistically
significant ability to predict malignant transformation in
oral dysplasia.23 The role of ghrelin in oral carcinogenesis
is not fully understood, but the ghrelin expression changes
in oral carcinogenesis may be a biomarker that can
improve the identification of increased malignant
potential. Further studies using larger sample sizes will
determine the value of ghrelin as a potential biomarker for
Acknowledgements: This work was supported by the
Scientific Research Project Fund of Cumhuriyet
University under project number DİŞ – 156.
Disclosure: None of the authors have any financial
interest either directly or indirectly in any company or any
of the products mentioned in this manuscript.
The English in this document has been checked by at least
two professional editors, both native speakers of English.
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Doç. Dr. İlker ÖZEÇ
Diş Hekimliği Fakültesi
Ağız, Diş ve Çene Cerrahisi AD