Material & methods

Study design and participants

This observational case–control study initially enrolled 45 participants (Table 1) categorized into three groups: healthy controls (n = 15), leukoplakia (n = 15), and oral squamous cell carcinoma (OSCC) (n = 15). Following real-time PCR quality control auditing, two samples within the healthy control cohort failed to yield detectable target or reference gene signals and were excluded from the downstream analysis. The final dataset comprised 43 participants: 13 healthy controls, 15 with leukoplakia, and 15 with OSCC.

Characteristic | Healthy Controls (n = 15) | Leukoplakia (n = 15) | OSCC (n = 15)
Number of participants, n | 15 | 15 | 15
Age range (years) | 24–69 | 25–66 | 25–64
Male, n (%) | 9 (60.0) | 13 (86.7) | 10 (66.7)
Female, n (%) | 6 (40.0) | 2 (13.3) | 5 (33.3)
Predominant habit | None | Smoking and chewing | Tobacco chewing
Predominant sites | NA | Buccal mucosa | Buccal mucosa > Vestibule > Tongue

Healthy controls had no oral lesions, history of OPMD, malignancy, or systemic disease. They were recruited consecutively from individuals attending the outpatient dental department for routine dental examinations or minor dental treatments. All controls underwent a comprehensive oral examination by an oral pathologist to confirm the absence of oral potentially malignant disorders, oral cancer, or any other oral mucosal lesions. Individuals with a history of OPMDs, malignancy, chronic systemic illness, or tobacco- or alcohol-related oral pathology were excluded. Participants with oral leukoplakia and oral squamous cell carcinoma were recruited based on clinical diagnoses recorded in the Department of Oral Medicine and Radiology. Individuals with systemic illnesses or prior cancer treatment were excluded. All patients included in these two groups had a tobacco-chewing habit. Histopathological confirmation of the study lesions was not performed as part of the clinical study protocol; therefore, clinicopathological correlations could not be assessed. Written informed consent was obtained from all participants prior to the sample collection.

The study was approved by the Institutional Ethics Committee (EC/NEW/INST/2021/2435- SL. NO. 287) and registered with the Clinical Trials Registry of India (CTRI/2024/08/072442 dated 13th August 2024).

Saliva collection

Unstimulated saliva (2 mL) was collected from participants who refrained from eating, drinking, or smoking for at least 30 min prior to collection. Samples were obtained by passive drooling into sterile containers and stored at −80 °C until further analysis.

DNA extraction

Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The DNA concentration and purity were assessed using a NanoPhotometer N60 (Implen, Munich, Germany).

Telomere length measurement by qPCR

Relative telomere length was assessed using a quantitative polymerase chain reaction (qPCR) method as described by Joglekar et al. (2020),14 with minor modifications related to instrumentation and reagents, while maintaining the original assay design and comparative ΔΔCt approach.

qPCR was performed using a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green PCR Master Mix (Green® Premix Ex Taq II; Takara Bio Inc., Shiga, Japan). Each reaction had a total volume of 10 μL and included primers specific for telomeric repeats or the β-globin single-copy reference gene.

The primer sequences used were as follows:•Telomere primers:oTel A (5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′)oTel B (5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′)•β-globin primers:ohbg1 (5′-GCTTCTGACACAACTGTGTTCACTAGC-3′)ohbg2 (5′-CACCAACTTCATCCACGTTCACC-3′)

The thermal cycling conditions included initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Amplification specificity was confirmed using a melt curve analysis. No-template controls (NTC) were included for both primer sets. Although fluorescent amplification curves were detected in the Telomere NTC wells due to clean primer-dimer formations characteristic of self-complementary telomeric repeat primers (Tm≈79°C), all reference gene NTC wells remained completely clean and undetermined. All clinical samples were processed on a single 96-well plate to minimize inter-run variations.

The qPCR workflow adhered to key Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines,15 including melt curve validation and use of controls; however, technical replicates and amplification efficiency analysis were not performed.

Data analysis

Ct values for telomere and β-globin reactions were obtained using StepOne software v2.3 with automatic baseline and threshold settings. Relative telomere length (RTL) was calculated using the comparative 2−ΔΔCt method as described by Joglekar et al. (2020).14 ΔΔCt was defined as the difference between the target telomere Ct value and the single-copy reference β-globin Ct value. To stabilize calculations against outliers, the baseline calibrator was set as the mathematical mean ΔCt of the healthy control cohort. Final values are expressed as the non-linear 2−ΔΔCt relative ratio, where higher numbers directly denote longer relative telomeric repeats.

Statistical analysis was performed using Python (SciPy v1.x and Pingouin). Relative telomere length (RTL) values were calculated for each sample using the standard non-linear formula 2−ΔΔCt. Descriptive statistics were expressed as median and interquartile range (IQR) for RTL, given its non-normal distribution, with the mean and standard deviation (SD) reported for reference. Two samples in the healthy control group had missing ΔΔCt values and were excluded listwise rather than being imputed. The normality of the RTL distribution within each group was assessed using the Shapiro-Wilk test, which was significant (p < 0.05) in all three groups, indicating a violation of the normality assumption required for one-way ANOVA. Homogeneity of variance was confirmed using Levene's test (p = 0.850). Given the non-normal distribution, differences in RTL among the three cohorts were analyzed using the non-parametric Kruskal-Wallis H test. Pairwise group comparisons were performed using the Mann-Whitney U test with Bonferroni correction for multiple comparisons. A two-tailed p-value < 0.05 was considered statistically significant.