Potentially malignant disorders (PMDs) represent an intermediate stage in oral carcinogenesis and are associated with a higher risk of malignant transformation compared to normal mucosa [1]. Among these, oral leukoplakia and oral lichen planus (OLP) are the most commonly encountered conditions. Oral leukoplakia is strongly associated with tobacco exposure and demonstrates varying degrees of epithelial dysplasia, which directly correlates with malignant potential [2-5]. Oral lichen planus, an immune-mediated condition, also exhibits a risk of malignant transformation, particularly in erosive and atrophic variants [6,7]. Telomerase is a ribonucleoprotein enzyme responsible for maintaining chromosomal stability. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is considered the key determinant of telomerase activity and is upregulated in premalignant and malignant conditions [8-10]. Increased hTERT expression enables cellular immortalization, a hallmark of cancer progression. Immunohistochemistry provides a reliable method for detecting and localizing hTERT expression within tissues. This study aims to evaluate hTERT expression in oral leukoplakia and OLP and compare it with normal mucosa.

Study Design and Setting This study was designed as a retrospective case–control study conducted in accordance with institutional ethical guidelines. The study was carried out using archived biopsy specimens obtained from the Department of Oral Pathology, Oral Medicine & Radiology, GITAM Dental College & Hospital, and the Department of Dermatology, GITAM Medical College & Hospital, Visakhapatnam. Study Population and Sample Size A total of 40 subjects were included in the study and categorized into three groups: • Group 1 (Control group): 10 healthy individuals without oral lesions or deleterious habits • Group 2 (Leukoplakia group): 15 patients clinically diagnosed with oral leukoplakia and histopathologically confirmed epithelial dysplasia • Group 3 (OLP group): 15 patients clinically and histopathologically diagnosed with oral lichen planus Inclusion Criteria • Healthy individuals free from oral lesions and systemic diseases • Untreated cases of oral leukoplakia with histopathologically confirmed epithelial dysplasia • Untreated cases of oral lichen planus with or without dysplastic changes Exclusion Criteria • Patients with systemic diseases or malignancies • Previously treated cases of leukoplakia or OLP • Clinically similar lesions such as candidiasis, pemphigus, pemphigoid, lupus erythematosus, and lichenoid reactions • Pregnant and lactating women • Individuals with deleterious habits in the control group Sample Collection and Processing After obtaining informed consent, a detailed clinical examination and case history were recorded for each subject. Incisional biopsy specimens were obtained under local anesthesia in aseptic conditions. Control tissues were collected from non-inflamed sites during routine dental procedures. [figure 1-4] All tissue specimens were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Sections of 4 µm thickness were prepared and mounted on polysilane-coated slides. Hematoxylin and eosin staining was performed to confirm the histopathological diagnosis. Immunohistochemical Procedure Immunohistochemical analysis was performed using a monoclonal antibody against human telomerase reverse transcriptase (hTERT). The staining procedure included the following steps: 1. Deparaffinization and Rehydration: Sections were dewaxed in xylene and rehydrated through graded alcohol. 2. Antigen Retrieval: Heat-induced epitope retrieval was performed using EDTA buffer (pH 9) in a controlled heating system. 3. Blocking: Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, followed by application of a protein blocking agent to prevent non-specific staining. 4. Primary Antibody Incubation: Sections were incubated with monoclonal hTERT antibody for 1 hour at room temperature. 5. Secondary Antibody: Polymer-based horseradish peroxidase-conjugated secondary antibody was applied. 6. Chromogen Development: Diaminobenzidine (DAB) was used as the chromogen to visualize antigen-antibody reaction. 7. Counterstaining: Slides were counterstained with hematoxylin, dehydrated, cleared, and mounted. Controls Testicular tissue was used as a positive control due to its known high telomerase activity, while normal oral mucosa served as a negative control. Sections treated without primary antibody were also used as negative controls. Evaluation of Immunostaining Qualitative Assessment Positive immunoreactivity was defined as brown staining in the nucleus and/or cytoplasm of epithelial cells. Cellular localization was categorized as: • Nuclear • Cytoplasmic • Combined nuclear and cytoplasmic Quantitative Assessment For each slide, ten representative high-power fields (400×) were selected. The following parameters were assessed: • Labeling Index (LI): Percentage of positively stained cells among total cells counted • Staining Intensity (SI): Graded as 0 (none), 1 (mild), 2 (moderate), 3 (intense) • Labeling Score (LS): Calculated as LI × SI Overexpression of hTERT was defined as staining in more than 10% of cells. To minimize observer bias, the evaluation was performed independently by multiple investigators, and average values were considered. Statistical Analysis Data were compiled and analyzed using descriptive statistical methods. Comparative evaluation between groups was performed, and results were interpreted based on observed differences in expression patterns. Figure 1 – H&E, IHC Stain in Control Figure 2 - Clinical case of Homogenous Leukoplakia, exhibiting Mild Epithelial Dysplasia in histopathology Figure 3 - Clinical case of Homogenous Leukoplakia, exhibiting Moderate Epithelial Dysplasia in histopathology Figure 4 – Clinical case of Speckled Leukoplakia, exhibiting Severe Epithelial Dysplasia in histopathology Figure 5 – hTERT positivity in variants of oral lichen planus

Table 1: Age Distribution among Study Groups The age distribution demonstrated distinct patterns across the study groups. In the control group, all subjects were below 40 years, with equal distribution in the 21–30 and 31–40 age categories. In contrast, patients with oral leukoplakia showed a higher prevalence in the 41–50 age group, accounting for the largest proportion of cases, followed by the 51–60 age group. Oral lichen planus (OLP) cases were predominantly observed in the 31–50 age range, with peak occurrence in both the 31–40 and 41–50 age groups. These findings indicate that leukoplakia tends to occur at a relatively later age compared to OLP, while both conditions are largely concentrated in the middle-aged population. Fig 1 Table 2: Clinical and Histopathological Grading of Leukoplakia Analysis of clinical variants of leukoplakia revealed a slightly higher prevalence of non-homogenous lesions compared to homogenous types. This is clinically significant, as non-homogenous leukoplakia is generally associated with a higher risk of malignant transformation. Histopathological grading showed an equal distribution of mild, moderate, and severe epithelial dysplasia among the cases. The uniform representation across dysplasia grades suggests that the study population included a balanced spectrum of disease severity, allowing for effective comparison of biomarker expression across different stages of epithelial alteration. Table 3: hTERT Localization Patterns A progressive alteration in hTERT localization was observed across the study groups. In normal oral mucosa, hTERT expression was confined exclusively to the nucleus, predominantly within the basal cell layer, indicating physiological telomerase activity. In leukoplakia, early dysplastic changes (mild epithelial dysplasia) also exhibited nuclear localization; however, with increasing severity, a shift toward combined nuclear and cytoplasmic staining became evident. Moderate dysplasia demonstrated mixed localization, while severe dysplasia showed complete transition to both nuclear and cytoplasmic expression. Fig 2 Similarly, in oral lichen planus, reticular and other less aggressive forms predominantly exhibited nuclear staining, whereas erosive variants demonstrated strong combined nuclear and cytoplasmic localization. This progressive change in staining pattern reflects increasing cellular dysregulation and proliferative activity. The findings suggest that cytoplasmic localization of hTERT may be associated with advanced dysplastic changes and higher malignant transformation potential. Table 1: Age Distribution among Study Groups Age Group Normal (%) Leukoplakia (%) OLP (%) 21–30 50 6.7 20 31–40 50 13.3 33.3 41–50 0 40 33.3 51–60 0 26.7 13.3 61–70 0 13.3 13.3 Table 2: Clinical and Histopathological Grading of Leukoplakia Parameter Category Percentage Clinical Homogenous 46.66 Clinical Non-homogenous 53.33 Histopathology Mild dysplasia 33.33 Histopathology Moderate dysplasia 33.33 Histopathology Severe dysplasia 33.33 Table 3: hTERT Localization Patterns Group Nuclear (%) Nuclear + Cytoplasmic (%) Normal 100 0 Mild Dysplasia 100 0 Moderate Dysplasia 60 40 Severe Dysplasia 0 100 Erosive OLP 0 100 Figure 1: Age distribution comparison across study groups Figure 2: hTERT localization patterns

Potentially malignant disorders (PMDs) of the oral cavity represent a critical stage in the multistep process of carcinogenesis, with oral leukoplakia and oral lichen planus (OLP) being among the most frequently encountered entities [1,4]. The present study evaluated the expression of human telomerase reverse transcriptase (hTERT) in these conditions and demonstrated a progressive increase in expression from normal mucosa to dysplastic lesions, supporting its role as a biomarker for malignant transformation. The demographic distribution observed in this study is consistent with established literature. Oral leukoplakia showed a higher prevalence in males, which can be attributed to increased exposure to deleterious habits such as tobacco consumption [5–7]. In contrast, OLP demonstrated a female predominance, reflecting its autoimmune etiology and association with systemic and psychological factors [8,9]. The age distribution, with peak incidence in middle-aged individuals, aligns with previous epidemiological studies reporting higher occurrence of PMDs in the fourth and fifth decades of life [4,5]. Buccal mucosa was identified as the most commonly affected site in both leukoplakia and OLP in the present study. This observation is in agreement with earlier reports, which attribute the high involvement of buccal mucosa to its frequent exposure to carcinogens, mechanical irritation, and chronic inflammatory stimuli [5,6]. The site predilection further emphasizes the importance of thorough clinical examination of high-risk areas in early detection strategies. One of the key findings of this study is the progressive increase in hTERT expression with the severity of epithelial dysplasia. In normal mucosa, hTERT expression was limited to basal cells, reflecting physiological cellular turnover and controlled proliferative activity. This is consistent with the understanding that telomerase activity is minimal in most somatic cells and is tightly regulated under normal conditions [10,11]. However, in leukoplakia and OLP, hTERT expression extended beyond the basal layer into suprabasal and superficial layers, indicating disruption of normal cellular regulatory mechanisms. Telomerase plays a fundamental role in maintaining chromosomal integrity by elongating telomeric DNA sequences. The catalytic subunit, hTERT, is considered the primary determinant of telomerase activity and is often upregulated in premalignant and malignant tissues [11–13]. Increased expression of hTERT enables cells to bypass replicative senescence and acquire unlimited proliferative potential, a hallmark of cancer development [12,13]. The findings of this study support this biological premise, as higher levels of hTERT expression were observed in lesions with greater dysplastic changes. A notable observation in this study was the shift in hTERT localization from nuclear to combined nuclear and cytoplasmic staining with increasing severity of dysplasia. In mild epithelial dysplasia, staining was predominantly nuclear, whereas moderate and severe dysplasia exhibited both nuclear and cytoplasmic localization. Similar patterns were observed in OLP, where erosive variants demonstrated more intense and widespread staining compared to reticular forms. This alteration in localization may reflect changes in protein transport, post-translational modifications, or intracellular signaling pathways associated with malignant transformation. Previous studies have also reported increased telomerase activity in PMDs and oral cancers. O’Flatharta et al. demonstrated telomerase activity in OLP using RNA in situ hybridization but concluded that its presence alone may not be sufficient as a marker for malignant transformation [13]. However, immunohistochemical detection of hTERT provides additional advantages by allowing localization of expression within specific cell layers, thereby improving diagnostic accuracy [14,15]. Palani et al. reported increased hTERT expression in oral precancer and cancer, supporting its role as a reliable biomarker [15]. The progressive increase in hTERT expression observed in this study, particularly in severe epithelial dysplasia and erosive OLP, suggests a direct correlation with malignant potential. This finding is clinically significant, as it may aid in identifying high-risk lesions that require closer monitoring or early intervention. Furthermore, the equal distribution of dysplasia grades in leukoplakia cases in this study provided a balanced framework to assess the relationship between biomarker expression and disease severity. The use of immunohistochemistry in this study offers practical advantages in routine diagnostic settings. Unlike molecular techniques such as the telomeric repeat amplification protocol (TRAP), immunohistochemistry allows visualization of protein expression within tissue architecture and is less susceptible to false-positive results due to lymphocyte contamination [13,14]. However, variability in staining intensity and interpretation remains a limitation, emphasizing the need for standardized protocols. Despite the valuable insights provided by this study, certain limitations must be acknowledged. The sample size was relatively small, which may limit the generalizability of the findings. Additionally, the cross-sectional design does not allow for long-term follow-up to directly correlate hTERT expression with actual malignant transformation. Future studies with larger sample sizes and longitudinal follow-up are required to validate these findings and establish definitive prognostic thresholds.

The present study demonstrates that hTERT expression increases progressively from normal mucosa to leukoplakia and oral lichen planus, correlating with the severity of epithelial dysplasia. The findings support the potential role of hTERT as a prognostic biomarker for malignant transformation in potentially malignant disorders. Further large-scale studies are required to validate its clinical applicability.

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