| Abstract|| |
Purpose: Telomerase is a specialized ribonucleoprotein complex that stabilizes telomeres by adding "TAG" repeats to the end of chromosomes. The catalytic subunit of telomerase is human telomerase reverse transcriptase (hTERT), whose expression is the critical determinant of telomerase activity. Telomeres and telomerases play an important role in the longevity of cell and are known to conform "immortalization" on neoplastic cells. Although there exists a lot of information on telomerase in oral cancer, very little is known about their expression in leukoplakia and oral submucous fibrosis (OSF). This study addresses this lacuna.
Materials and Methods: In this preliminary study, immunohistochemistry (IHC) was used to detect the expression of hTERT protein in oral squamous cell carcinoma (OSCC) (n=30), leukoplakia (n=15), OSF (n=15) and normal oral mucosa (n=10). The cellular localization of immunostain, intensity of stain, mean nuclear labeling index (LI) and mean nuclear labeling score (LS) of hTERT protein were studied. A total number of 1000 cells were counted in each slide. All the data were analyzed using SPSS software version 10.0.2. The cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity and LI were compared across the groups using Pearson's χ2 test. The mean LI and LS for OSF, leukoplakia, OSCC and normal were compared using analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant.
Results: The mean nuclear LI increased from OSF (22.46±4.53), through normal (28.3±12.3) to OSCC (47.56±21.30) (P=0.002) and from normal (28.3±12.3), through leukoplakia (44.06±14.6), to OSCC (47.56±21.30) (P=0.00). The mean nuclear labeling score was observed to increase from OSF (37.8±15), through normal (64.9±30.7), to OSCC samples (106.9±29.77) (P=0.00) and from normal (64.9±30.7), through leukoplakia (85.6±25.1) to OSCC samples (106.9±29.77) (P=0.00).
Conclusion: There was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. The cellular localization, LI and LS in OSF were significantly different from OSCC and leukoplakia.
Keywords: Cancer, leukoplakia, oral, oral submucous fibrosis, telomerase
|How to cite this article:|
Palani J, Lakshminarayanan V, Kannan R. Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer. Indian J Dent Res 2011;22:362-3
Oral cancer is the eighth most common cancer world-wide and the third most common cancers in the South-East and Central Asian region. In India, the incidence rate of oral cancer is reported at 12.6% per 1,00,000 population.  Oral squamous cell carcinoma (OSCC) is often preceded by a distinct, clinically identifiable potentially malignant lesion/condition, such as leukoplakia or oral submucous fibrosis (OSF). ,
|How to cite this URL:|
Palani J, Lakshminarayanan V, Kannan R. Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer. Indian J Dent Res [serial online] 2011 [cited 2014 Dec 19];22:362-3. Available from: http://www.ijdr.in/text.asp?2011/22/2/362/84281
Malignant transformation of normal mucosa is associated with genetic changes that affect cell cycle, apoptosis, angiogenesis and telomere length. Telomeres are highly conserved non-coding portions of DNA, which are made of tandem repeats of 'Thymine-Adenine-Guanine (TAG)' sequences. Maintenance of the telomeric length confers immortality to cells, which is a characteristic feature of malignant cells and is considered to be a critical step in carcinogenesis. 
A specialized ribonucleoprotein complex called telomerase stabilizes telomeres by addition of 'TAG' repeats to the ends of chromosomes. The telomerase complex consists of RNA template (hTR), a catalytic sub-unit called human Telomerase Reverse Transcriptase (hTERT) and associated protein (hTP-1).  hTERT expression (molecular weight ~130 kDa) is one of the critical determinants of telomerase activity. ,,,
Telomerase activity is up regulated in many tumors and studies have shown that increased telomerase expression is an early event in oral carcinogenesis. ,,,, Telomerase activity has been demonstrated in oral cancer and leukoplakia, but not much is known of its expression in OSF. ,,,,,,, The aim of our study was to ascertain the expression of hTERT as an indicator of telomerase activity in OSF, leukoplakia and OSCC using immunohistochemistry on formalin-fixed paraffin-embedded tissues.
| Materials and Methods|| |
The study samples obtained from archival biopsy specimens from the Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, Chennai comprised of OSF (n=15), leukoplakia (n=15) and OSCC (n=30). Histopathologically confirmed cases of OSCC, leukoplakia showing epithelial dysplasia and OSF showing dense fibrous connective tissue with epithelial atrophy were included in the study. Site-matched normal control tissues were obtained from non-inflamed buccal mucosa during removal of impacted third molar of patients attending the hospital. Informed consent was obtained from all subjects enrolled in the study and the study protocol was approved by the Institutional Review Board. All incisional biopsies were performed under local anesthesia (2% lignocaine). Tissues were fixed in 10% buffered formalin and paraffin embedded.
Immunohistochemical detection of hTERT protein
Five micrometer sections were obtained from paraffin-embedded tissues and were placed on 3-amino propyl triethoxy silane (APES) coated slides. The sections were deparaffinized and brought to water. The sections were placed in sodium citrate buffer (pH 6) and antigen retrieval was done using an autoclave at 15 psi for 15 minutes. The slides were then washed in cold Tris Buffer Saline (TBS) (pH 7.2); three changes of TBS washes were performed after every step during the immunostaining procedure. Peroxidase and protein blocking were performed with 3% hydrogen peroxide and protein block provided in the secondary kit. , The sections were incubated with mouse anti-human telomerase reverse transcriptase monoclonal primary antibody (NCL-hTERT, Novocastra Laboratories?, New York, USA) at 1:40 dilution at room temperature for 1 hour. The secondary antibody (Novocastra Peroxidase Detection system) used was biotinylated goat anti-mouse immunoglobulin (30 minutes at room temperature), followed by addition of streptavidin - peroxidase conjugate (30 minutes at room temperature). The sections were then treated with diaminobenzidine hydrochloride for 5 minutes, counterstained with hematoxylin and mounted with DPX® . Tissue obtained from tonsils during tonsillectomy was used as positive control  and sections treated with TBS in the place of primary antibody served as negative controls.
Hematoxylin and eosin-stained sections were used to confirm clinical diagnosis and grade the specimens. OSCC samples were graded as well, moderately or poorly differentiated based on established criteria.  Leukoplakia samples were histopathologically graded as mild, moderate or severely dysplastic.
hTERT expression in the epithelium was assessed as described by Luzar et al.  The parameters studied were as follows:
- Cellular localization of the stain was defined as being either nuclear/cytoplasmic/both.
- The staining intensity (SI) of the samples were graded and assigned numerical scores (0, 1, 2, 3 for no stain, mild, moderate and intense stain respectively). Three investigators performed the assessment independently and each investigator used the positive control as standard bench mark. The inter-observer correlations were 0.63 and 0.62.
- Nuclear labeling indices (LI) were calculated as percentage of hTERT-stained cells per thousand cells counted. The counting was done using eye piece graticule under high power objective (40x).
- The nuclear labeling scores (LS) of the samples were determined using the formula LI × SI.  To eliminate the bias in labeling score (LS), five different sites were counted by two different examiners.
The SI, LI and cellular localization of hTERT stain were compared across the groups using Pearson's χ2 test. Kappa statistics was performed for three observers to evaluate the inter-observer variability for the intensity of stain and hTERT signal. The mean LI and LS for OSF, leukoplakia, OSCC and normal were compared using analysis of variance (ANOVA), while Student t-test was used for comparison between two groups. The analyses were performed using Statistical Package for Social Sciences (SPSS) version 10.0.2 and P-value less than 0.05 was considered statistically significant.
| Results|| |
The demographic details of all the patients included in the study are shown in [Table 1]. OSCC cases comprised of 50% (15/30) well differentiated, 33% (10/30) moderately differentiated and 17% (5/30) poorly differentiated carcinomas. Of the 15 cases of leukoplakia, 33% (5/15) of the samples exhibited mild, 46.7% (7/15) moderate and 20% (3/15) severe dysplasia.
hTERT staining characteristics
Nuclear staining was observed in 36.7% (11/30) and combined nuclear and cytoplasmic staining in 63.3% (19/30) samples [Figure 1]a. Immuno-staining intensity analysis showed that 46.7% (14/30) exhibited moderate staining, 33.3% (10/30) had mild staining and 20% (6/30) intense staining. The cellular distribution and intensity of hTERT stain did not show significant difference with differentiation (P=0.93) (P=0.82). The LI and LS for OSCC were 47.56±21.30 (95% CI=39.9-55.1) and 106.9±29.77 (95% CI=96.2-117.5) respectively. LI and LS of hTERT stain did not show any significant correlation across the degree of differentiation.
|Figure 1: Expression of hTERT protein in (a) oral squamous cell carcinoma (b) leukoplakia (c) oral submucous fibrosis and (d) normal oral mucosa|
Click here to view
73.3% (11/15) exhibited only nuclear staining and 26.7% (4/15) of the samples showed nuclear and cytoplasmic staining. Fifty-three percentage (8/15) of the cases had mild staining [Figure 1]b and 20% (3/15) moderate staining, while 26.7% (4/15) exhibited intense staining. The cellular localization and intensity of hTERT stain across the grades of dysplasia were not statistically significant. The LI and LS for leukoplakia were 44.06±14.6 and 85.6±25.1 respectively. The mean LI across the grades of dysplasia did not reveal any statistical significance. We observed that the increase in mean nuclear LS from mild (60.2±20.9), to moderate (93.8±17.2) to severe (108.6±3.21) dysplasia was statistically significant (P=0.004).
Nuclear staining was seen in 86.7% (13/15) and combined nuclear and cytoplasmic staining in 13.3% (2/15) [Figure 1]c. Equal number of cases (5) exhibited mild, moderate and intense staining. The LI and LS for OSF were 22.46±4.53 and 37.8±15.
Eighty percent (8/10) showed predominantly nuclear staining and one specimen had combined nuclear and cytoplasmic staining. Fifty percentage (5/10) of the samples showed mild staining [Figure 1]d, 30% (3/10) moderate, 10% (1/10) intense staining, and 10% (1/10) exhibited no staining. The normal controls exhibited a nuclear LI and LS of 28.3±12.3 and 64.9±30.7 respectively.
Comparison between groups
The difference in the cellular localization of hTERT immunostain in OSF, leukoplakia, OSCC and normal was statistically significant (P=0.001) [Figure 2]a. The comparison of cellular localization of hTERT stain between OSCC and leukoplakia (P=0.020), OSCC and OSF (P=0.002) and OSCC and normal (P=0.006) revealed statistically significant differences between the lesions. Although there were differences in the SI across the groups, the differences were not statistically significant [Figure 2]b.
|Figure 2: Comparison of cellular localization, intensity, labeling score and labeling index across the study groups|
Click here to view
The mean nuclear LI increased from OSF (22.46±4.53), through normal (28.3±12.3) to OSCC (47.56±21.30) (P=0.002) [Figure 2]c. The mean LI was higher in OSCC when compared to OSF and normal samples and the difference was statistically significant (P=0.000; P=0.010). The mean LI increased from normal (28.3±12.3), through leukoplakia (44.06±14.6), to OSCC (47.56±21.30) (P=0.00) [Figure 2]c. The mean LI was significantly higher in OSCC when compared to leukoplakia (P=0.00).
The mean nuclear LS increased from OSF (37.8±15), through normal (64.9±30.7), to OSCC samples (106.9±29.77) [Figure 2]d. The difference in the mean nuclear LS among the groups was statistically significant (P=0.000). A significant higher mean nuclear LS was observed in OSCC when compared to OSF (P=0.000) and normal controls (P=0.007). The mean nuclear LS increased from normal (64.9±30.7), through leukoplakia (85.6±25.1) to OSCC samples (106.9±29.77) (P=0.00) [Figure 2]d. A significant higher mean nuclear LS was observed in OSCC when compared to leukoplakia (P=0.02).
| Discussion|| |
hTERT is a nuclear protein which converts the RNA template (hTR) into a DNA sequence and thus serves to elongate or maintain telomere length.  Expression of hTERT is considered to be the critical determinant of telomerase activity in neoplasias, as hTR and hTP-1 are ubiquitously expressed by both normal and cancerous cells, whereas an increased hTERT expression is observed predominantly in cancerous cells. , The conventional method of studying telomerase activity - Telomerase Repeat Amplification Protocol (TRAP) does not illustrate cellular localization, which is possible with hTERT IHC.  In the present study, immunohistochemical expression of hTERT protein was analysed and compared among normal, potentially malignant disorders and OSCC samples.
In OSF, leukoplakia and normal tissues, a significant nuclear staining was observed, while greater proportion of OSCC exhibited combined nuclear and cytoplasmic staining (P=0.001). In the present study, only one sample of normal oral mucosa had cytoplasmic staining, while in OSCC, leukoplakia and OSF tissues, hTERT cytoplasmic expression ranged from 13-33%. The 10% of normal cases showing hTERT staining might be due to the molecular changes related to malignant conversion that may be happening in these sites. The telomerase expression in normal mucosa has to be further elaborated by TRAP due to limitations in IHC technique. The cytoplasmic localization of hTERT staining has been previously reported in carcinomas of breast, cervix and larynx, which represents unphosphorylated and inactive forms of the protein, probably awaiting nuclear translocation. ,, The implication of the cytoplasmic expression of hTERT in some cases and its correlation to behavior is an area of active research.
Although differences in intensity of staining were seen, the inter-group or intra-group differences of hTERT SI were not statistically significant.
In this study, increasing LI and LS were observed from normal through leukoplakia with dysplasia to OSCC implying that hTERT expression increases during malignant transformation. Yajima et al. Mao et al. and Fujimoto et al. have observed a similar increase in telomerase activity from normal through leukoplakia to oral cancer. ,, Studies of hTERT expression in laryngeal carcinogenesis have shown increased nuclear staining from normal through epithelial dysplasia to laryngeal squamous cell carcinoma. 
There was significant decrease in the nuclear LI and LS of OSF when compared with normal, leukoplakia and OSCC in our study. Chang et al. and Patel et al. reported telomerase activity in 40% (2/5 cases) and 100% (4/4 cases) of OSF samples respectively. , Chang et al. suggest that weak telomerase activity of OSF in their study might be due to contamination of inflammatory cells from the underlying fibrous connective tissue.  Further, TRAP assay was used to detect the telomerase activity in these studies and hence LI and LS were not calculated.
In OSF, the primary pathology is the abnormal progressive fibrosis of the connective tissue and subsequent epithelial atrophy.  Malignant transformation of OSF is frequently associated with dysplasia of the overlying epithelium.  All the cases of OSF in this study exhibited epithelial atrophy, but no dysplastic features were identified. The decrease in the thickness of epithelium is associated with the decrease of the stem cell or transition cell populations of the basal layer  and probably accounts for the low expression of nuclear LI and LS among the OSF samples.
In the current study, there was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. hTERT immunostain parameters (cellular localization, nuclear LI and nuclear LS) in OSF were significantly different from OSCC and leukoplakia. The localization of hTERT stain in OSF was predominantly nuclear, whereas in leukoplakia and OSCC, more proportion of cells had both nuclear and cytoplasmic staining. The LI and LS gradually increased from OSF through normal to leukoplakia to OSCC. In OSF, the number of cells expressing altered hTERT expression was less. This could imply two things: one, there is decreased telomerase activity or these cells are using ALT (Alternative Lengthening of Telomeres) mechanisms to maintain the telomere length.  These mechanisms have to be explored by further extensive studies using TRAP to establish immunohistochemical detection of telomerase as a potential marker of malignant transformation.
The present study characterizes hTERT in OSF by IHC and discusses its implications and directions for further studies. This is one of the preliminary studies of hTERT expression in OSF and further work is ongoing in the department.
| Ackowledgments|| |
We thank our Principal, Dr. S. Ramachandran, for encouraging and facilitating the publication of this article. We also thank our staff members Dr. M. Umadevi and Dr. Elizabeth Joshua for providing guidance and for proof reading the article.
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Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, Chennai, Tamil Nadu
[Figure 1], [Figure 2]