Oral cancer is the sixth most common malignancy

worldwide (1). The incidence of oral cancer in the

world is much higher in South and South-East Asia
with more than 100,000 cases occurring every year
with poor survival rate (2). The major predisposing
factors to oral cancer in this region are tobacco smok-
ing and betel quid chewing (3,4).
Oral carcinogenesis is a multi-step process involv-
ing distinct genetic alterations. Chromosomal losses
have been shown to occur at each histopathological
step from benign hyperplasia to dysplasia to carcino-
ma in situ to invasive head and neck cancers (5). In
addition, clinically recognizable precursor lesions to
oral carcinoma such as leukoplakia and erythroplakia
occasionally occur. They are referred to as potential-
ly malignant oral lesions (6). Histologic criteria used
to predict the malignant potential of these lesions is
termed epithelial dysplasia (7). The traditional
histopathological diagnosis of potentially malignant
oral lesions using dysplasia criteria has been shown to
be highly subjective (8,9). The need for molecular
markers to be used as an adjunct to histopathological
assessment in predicting which potentially malignant
oral lesion will transform to oral cancer has been well
recognized (10).
The checkpoint mechanisms that limit the replica-
J. Exp. Clin. Cancer Res., 24, 4, 2005
639
Human Telomerase Reverse Transcriptase Expression in Oral
Carcinogenesis - A Preliminary Report
S.K.S. Kumar
1
, R.B. Zain
1
, S.M. Ismail
2
, S.C. Cheong
3
Dept. of Oral Pathology, Oral Medicine and Periodontology
1
, Dept. of Oral and Maxillofacial Surgery
2
, Faculty of Dentistry, University
of Malaya, Kuala Lumpur; Cancer Research Initiatives Foundation (CARIF)
3
, Outpatient Centre, Subang Jaya Medical Centre, Subang
Jaya, Selangor Darul Ehsan; Malaysia
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is strongly associated with
telomerase activity implicated in cellular immortalization and tumorigenesis. In situ detection of hTERT will aid
in determining the localization of telomerase positive cells. The aim of this study was to detect hTERT protein
expression in multistep oral carcinogenesis using paraffin embedded tissue samples, and to study the relationship
of hTERT expression with different histological stages in oral carcinogenesis. Normal (n=4), hyperplastic (n=4),
dysplastic (n=4) and neoplastic (n=10) oral epithelia representing different histological stages in oral carcino-
genesis were included in the study. hTERT protein detection was done by immunohistochemistry (IHC) tech-
nique. Nuclear staining intensities were noted and the hTERT-labelling index was determined. Dysplastic and
neoplastic oral epithelia showed an increased percentage of hTERT positive cells (Grade 4: >50% positive stain-
ing nuclei) with intense staining in the basal, parabasal and superficial layers of the epithelia, unlike normal oral
mucosa which showed intense staining only in the basal and parabasal cell layers, which are the normal prolifer-
ative progenitor compartments. hTERT protein expression was elevated with the corresponding advancement of
the histological stages of oral carcinogenesis, from normal to hyperplasia to dysplasia to carcinoma. There seems
to be an upregulation of hTERT protein expression during the progression of oral cancer, therefore, this may in-
dicate the feasibility of IHC detection of hTERT protein in oral carcinogenesis as a potential diagnostic or prog-
nostic marker.
Key Words: Telomerase, Human telomerase reverse transcriptase, Oral squamous cell carcinoma, Oral
precancer, Immunohistochemistry
tive life span of normal cells such as the progressive
shortening of telomeres, are altered in cancer (11).
Telomeres are DNA-protein structures at the ends of
linear chromosomes. They aid chromosome stabiliza-
tion and replication. In normal cells, telomeres short-
en with each cell division because of the end-replica-
tion problem which is the inability of the lagging
strand of DNA synthesis to replicate the 3' end of the
chromosome (12). They shorten until reaching a cer-
tain length beyond which cells no longer replicate and
undergo senescence. Occasionally, cells with critical
telomere length bypass the senescence barrier and
undergo chromosomal instability that initiates the
acquisition of telomere maintenance mechanisms,
namely the activation of telomerase and alternative
lengthening of telomere (ALT) mechanisms to main-
tain stable telomere lengths. This stabilization per-
mits immortalization and tumorigenesis (13,14).
Telomerase is a ribonucleoprotein complex syn-
thesizing TTAGGG repeats (15) with core units of
human telomerase RNA (hTR) (16) and human
telomerase reverse transcriptase (hTERT) (17).
Telomerase activity (TA) is found in stem cells, germ
cells and tumor cells but not in the majority of somat-
ic cells. TA is usually repressed in somatic cells with
a limited lifespan. Infinite proliferation capacity or
immortality is achieved by telomerase activation
(18). Telomerase activation is detected in about 85%
of a variety of tumor samples using various molecu-
lar techniques such as PCR-based Telomeric Repeat
Amplification Protocol (TRAP) assay and RT-PCR to
detect hTERT mRNA and hTR (19,20). A major
drawback in using these techniques is false positives
obtained due to the presence of normal physiological-
ly renewing cells such as lymphocytes and proliferat-
ing progenitor stem cells in the tumor tissue analysed
(21-23). Given this, the use of in situ detection of TA
using immunohistochemistry methods is more accu-
rate as it allows the direct visualization of where the
telomerase positive cell types are localised (24).
Human telomerase reverse transcriptase (hTERT)
is the catalytic subunit and rate-limiting component
of telomerase enzyme complex activity (17,25-27).
Strong correlation has been observed between hTERT
mRNA and/or protein expression and TA in a variety
of malignant tumors (28-33).
The detection of telomerase activity in oral pre-
cancer and cancer tissues has been done predomi-
nantly by TRAP assays (31,34-36). TA has also been
detected from oral rinses of patients with head and
neck squamous cell carcinoma using the TRAP assay
(21). Only few in situ studies have been attempted in
oral tissues with in situ RT-PCR (11), in situ
hybridization (29) and immunohistochemistry (37).
The potential of immunohistochemical detection of
telomerase in potentially malignant oral lesions as a
biomarker for high-risk lesions has been recognized
(38). The only study published on immunohistochem-
ical detection of hTERT in oral carcinogenesis (37)
used dysplastic and neoplastic tissue samples but did
not report on the pattern of hTERT protein expression
in normal and hyperplastic oral epithelia. In addition,
no study has been reported on hTERT protein expres-
sion in oral cancer in Malaysia.
The objectives of this study are to detect hTERT
protein expression using immunohistochemistry in
different histological stages (normal, hyperplasia,
dysplasia and squamous cell carcinoma) of oral car-
cinogenesis using paraffin embedded tissue samples,
and to study the relationship of hTERT expression
with different histological stages in oral carcinogene-
sis.
Materials and Methods
Oral squamous cell carcinoma (n=10), epithelial
dysplasia (n=4, potentially malignant oral lesions),
epithelial hyperplasia (n=4) and normal oral mucosa
samples (n=4) obtained from an archival specimen
collection were included in the study. All epithelial
hyperplasia and dysplasia tissues were from patients
clinically diagnosed as leukoplakia. The samples
were fixed in 10% buffered neutral formalin and
embedded in paraffin. Tissue sections (4m thick-
ness), were made for histological and immunohisto-
chemical interpretation. Furthermore, final histologi-
cal interpretation was made by RBZ.
Two cultured cell lines, fibroblasts (immortalised
with hTERT) and oral keratinocytes (OSC-20: oral
squamous cell carcinoma cell line, JCRB 0197,
HSRRB, Japan) were used as positive controls. The
cells were tested for telomerase activity with PCR
based TRAP assay using commercially available
TRAPeze

kit (Intergen Company, USA). These cells

were also used to prepare cell blocks. Briefly, the
cells were fixed in 10% buffered neutral formalin,
resuspended in molten agarose and then embedded in
paraffin. Sections from these cell blocks were used as
positive procedural control in immunohistochemistry.
The negative control was obtained by replacing the
primary antibody with tris-buffered saline (TBS).
Immunohistochemistry was performed using the
Avidin-Biotin Complex/Horse Radish Peroxidase
S.K.S. Kumar et al.
640
method. Tissue sections were stained for hTERT
using a commercial antibody (NCL-L-hTERT, Novo-
castra, UK). Sections were dewaxed in xylene and
rehydrated in graded ethanol. Antigen retrieval was
done by incubating sections immersed in 0.01M cit-
rate buffer at pH 6.0, in a microwave oven at 99C for
20 minutes. The sections were allowed to cool down
at room temperature for 20 minutes. The sections
were then immersed in 1% hydrogen peroxide (H
2
O
2
)
in methanol for 20 minutes to block endogenous per-
oxidase activity. Following that, the sections were
washed in TBS (Tris-buffered saline, pH 7.6) before
being incubated in normal rabbit serum (1:5 dilution
with TBS) for 20 minutes to block non-specific bind-
ing. After draining off the excess serum, the sections
were incubated in the primary antibody (NCL-L-
hTERT, NovoCastra) for 2 hours at room tempera-
ture. The sections were washed in TBS before being
incubated with the secondary antibody (Biotinylated
Rabbit Anti-Mouse, DAKO; 1:300 dilution with
TBS) for 30 minutes. The sections were then washed
again with TBS and incubated with Avidin Biotin
Complex/Horseradish peroxidase for 30 minutes.
After washing with TBS, peroxidase activity was
visualized with light microscopy by applying DAB
(Diaminobenzidine tetrahydrochloride, DAKO) chro-
mogen. The sections were counterstained with
haematoxylin, dehydrated in increasing grades of
alcohol and finally mounted in dibutyl phthalate
(DPX) mountant.
Criteria for immunohistochemical staining inter-
pretation. For the purpose of interpretation of hTERT
staining in normal oral mucosa, hyperplasia and dys-
plasia, the oral epithelium was divided into 3 layers
as previously published (39): basal layer (cells in
contact with the basement membrane), parabasal
layer (two rows of cells above the basal layer) and
superficial layer (all cell layers towards the surface
from the parabasal layer). The basal and parabasal
cell layers of stratified squamous epithelium are the
proliferative progenitor compartments and mainly
contain the physiologically renewing stem cells. Cells
in the superficial layers are normally terminally dif-
ferentiated and are non-proliferating (40). The sec-
tions of oral squamous cell carcinoma were evaluated
at three different fields which showed numerous
tumor cells.
A homogenous staining or a speckled/dotted pat-
tern in the nucleus was considered a positive staining,
and the absence of distinct nuclear staining was taken
as negative staining. The grading for percentage of
stained cells (hTERT-labelling index) was made by
following previously published criteria (41) as fol-
lows: Grade 1: Negative staining; Grade 2: 1-10%
positive staining nuclei; Grade 3: 11-50% positive
nuclei; and Grade 4: >50% positive nuclei. Cell
counting in normal mucosa, hyperplasia and dyspla-
sia was done by observing tissue sections in three dif-
ferent fields in each subdivided epithelial compart-
ment (basal, parabasal and superficial layers) under
100X magnification using an image analyser (Image-
Pro

). The average count calculated from the readings

obtained in the three different fields in each subdivid-
ed epithelial compartment was considered to obtain
the hTERT labelling index. Cell counting in carcino-
ma sections were made in three different fields which
showed numerous tumor cells. The average count cal-
culated from the readings obtained in the three differ-
ent fields of carcinoma was considered to obtain the
hTERT labelling index. The intensity of positive
staining was defined as follows: strong/distinct dark
brown homogenous staining or a speckled/dotted pat-
tern in the nucleus and weak/faint light brown
homogenous staining or a speckled/dotted pattern in
the nucleus.
Results
The cultured cells (fibroblasts immortalised by
hTERT and oral squamous cell carcinoma cell line:
OSC-20) which were tested for telomerase activity
using TRAP assay gave positive results with all pro-
cedural controls. Strong nuclear staining was
observed in sections of the oral squamous cell carci-
noma cell line (OSC-20) block as seen in Figure 1a.
The negative control did not show nuclear staining or
any non-specific staining (Fig.1b). The basal and
parabasal layers in general showed intense hTERT
staining in all the tissue sections including dysplastic
and neoplastic epithelia. Normal and hyperplastic
mucosa showed higher grades (Grades 3 & 4) of
hTERT expression in the basal and parabasal layers,
which are the normal proliferative progenitor com-
partments and contain stem cells which normally
have hTERT or telomerase activity. Interestingly, we
observed weak intensity of staining in superficial lay-
ers of normal (Fig.2a) and hyperplastic mucosa. The
stromal lymphocytes were also stained positive which
was reported to express telomerase (22). The four
dysplastic specimens used were diagnosed histologi-
cally as moderate dysplasia. Two cases of the dys-
plastic oral epithelia showed Grade 4 staining with
hTERT in Oral Carcinogenesis
641
strong intensity in the superficial layers (Fig.2b). All
ten cases (100%) of squamous cell carcinoma sec-
tions showed Grade 4 hTERT staining (Fig.2c; Table
I). The tumor cells in the invading epithelium and the
stromal lymphocytes ubiquitously expressed hTERT
protein. In general, hTERT expression was elevated
with the corresponding advancement of the histologi-
cal stages of oral carcinogenesis, from normal to
hyperplasia to dysplasia to carcinoma.
Discussion
The staining pattern of hTERT expression was
seen as distinct staining localized in the nucleus and
there was an absence of non-specific staining. To dif-
ferentiate between the normal proliferating cells at
the basal and parabasal layers which has been shown
to express hTERT in normal physiological conditions
using other in situ techniques (22,23), and the abnor-
mally proliferating mitotic keratinocytes at the super-
ficial layers, we evaluated our results in normal oral
mucosa, hyperplasia and dysplasia by dividing the
epithelium into three basic compartments.
Tumor cells, progenitor stem cells at the basal and
parabasal layers, stromal and infiltrating lymphocytes
showed strong and distinct hTERT protein expres-
sion. The antibody used in this study is a monoclonal
antibody which increases the specificity towards the
hTERT antigen. hTERT protein expression in basal &
parabasal cells reveals the presence of normal prolif-
erating cells which constantly renew the oral mucos-
al epithelium. Expression of hTERT in superficial
layers where cells normally undergo differentiation
suggests telomerase activity, and this may indicate
further malignant transformation. The finding of
hTERT positive staining in stem cells and lympho-
cytes highlights the reason for ambiguous results
obtained by molecular studies done in whole tissues
such as the TRAP assay.
This study indicates that hTERT expression is
upregulated with increasing histological grades of
oral carcinogenesis. Earlier studies using normal,
benign and neoplastic tissues of colon (42,43), breast,
lung, skin, stomach, pancreas (24) have shown simi-
lar elevated expression of hTERT protein with
increasing histological grades in carcinogenesis. It
has also been shown that hTERT expression is upreg-
ulated with the degree of undifferentiation in tumor
tissues of the liver (30). The results of this current
study are similar to the findings of a previous study
by Nguyen et al. on oral dysplastic lesions and squa-
mous cell carcinomas of tumors derived from the
oropharynx region (37) where hTERT protein expres-
sion was upregulated with increasing histological
severity of oral carcinogenesis. However, this current
study also included the evaluation of hTERT expres-
sion in normal and hyperplastic oral lesions in which
S.K.S. Kumar et al.
642
Fig. 1 - a) hTERT detection in a section of oral squamous cell carcinoma cell block prepared with OSC-20 cell line, HSRRB, Japan. Note
the strong/distinct brown staining localized in the nucleus (arrows) (Original magnification: X400) b) No staining was observed
when the primary antibody was replaced with neutral buffer solution (TBS) (arrows). (Original magnification: X400)
A B
weak hTERT expression in superficial layers of nor-
mal and hyperplastic epithelia was observed. The pat-
tern of hTERT expression in normal epithelia has
been observed in colorectal tissue where the authors
postulated that these differentiation committed cells
might contain degraded or dormant proteins that
retain the hTERT antigenicity without functionally
active telomerase (42). Fujimoto et al. described
hTERT, hTR and hTEP1 mRNA localization in oral
tissues (29). They found that these components were
expressed not only in the basal layers of normal
epithelium but were also seen in superficial layers of
hyperplastic and hyperkeratotic leukoplakia tissue, a
pattern similar to hTERT protein expression that was
observed in this study. Down regulation of telomerase
activity in vitro has also shown to induce senescence
of oral keratinocytes (44) indicating that hTERT
plays a critical role in senescence and tumor initiation
in the oral squamous epithelium. The precise
sequence of hTERT down regulation in normal
mucosa during differentiation and senescence, and
upregulation of hTERT in precancerous oral mucosa
is not known. The hTERT upregulation observed in
differentiation committed cells in precancerous oral
mucosa may cause the reactivation of telomerase
enzyme complex which in turn will lead to cellular
immortalization and ultimately result in development
of oral cancer. The understanding of the biological
significance of hTERT expression and telomerase
activation in physiologically renewing cells and dif-
ferentiation committed cells will form the basis for
understanding the distortion of normal growth mech-
anisms in carcinogenesis. In situ studies on hTERT
expression could give important clues towards this
direction.
Conclusion:
There appears to be an upregulation of hTERT
hTERT in Oral Carcinogenesis
643
Fig. 2 - Immunohistochemical detection of hTERT antigen in a)
normal oral mucosa. Note the weak/faint hTERT staining
in cells in basal and parabasal layers (arrows) (Original
magnification: X200) b) dysplastic oral mucosa. Note the
hTERT positive staining seen in the superficial layers of
the dysplastic epithelium (arrows) (Original magnifica-
tion: X100) c) Oral squamous cell carcinoma. The tumor
cells ubiquitously expressed hTERT (arrows). hTERT ex-
pression was also observed in stromal lymphocytes
(Original magnification: X200).
A
B
C
protein expression with histological progression of
oral cancer. Elevated expression of hTERT/TAoccurs
early in human oral carcinogenesis as hTERT protein
expression was seen in superficial layers of dyplastic
oral epithelia. Although hTERT and telomerase show
much promise as tumor markers for early diagnosis of
oral cancers, the physiological significance and
sequence of hTERT up regulation in tumor initiation
need to be understood for its diagnostic or prognostic
significance. This is a preliminary attempt at detect-
ing human telomerase reverse transcriptase (hTERT)
using immunohistochemistry with a monoclonal anti-
body on paraffin embedded samples representing
multistage oral carcinogenesis. The biological signif-
icance of hTERT expression in differentiation com-
mitted and proliferating cells will be analyzed in
future studies in order to elucidate if hTERT may be
a useful diagnostic or prognostic marker in oral can-
cer.
Acknowledgements: The authors would like to
thank Mrs. Rusnani Kamal for her expert technical
assistance. This study was presented at the Fourth
Scientific Meeting of IADR (International Associa-
tion for Dental Research), Malaysian Section, South
East Asian Division on 21 February 2004 at Univer-
sity of Malaya, Kuala Lumpur and the presenting
author (SK) was awarded the IADR Young Investiga-
tor Travel Award.
This study was supported by research grant (Vote
F 0213/2003A) of University of Malaya & in part by
IRPARMK-8 grant 06-02-03-0174 PR 0054/05-05 of
MOSTI, Malaysia.
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Received: April 27, 2005
Dr. Satish Kumar Shyam Kumar, BDS
Department of Oral Pathology, Oral Medicine and
Periodontology,
Faculty of Dentistry, University of Malaya,
Kuala Lumpur 50603, Malaysia.
Tel.: 00-603-79674803/ 4569.; Fax: 00-603-79674531
E-mail: satishkumarshyamkumar@yahoo.com
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