Result

A total of 45 participants were included in the study, comprising 15 healthy controls (9 males, 6 females; age range 24–69 years), 15 patients with leukoplakia (13 males, 2 females; age range 25–66 years), and 15 patients with oral squamous cell carcinoma (OSCC) (10 males, 5 females; age range 25–64 years) (Fig. 1) (Table 1). The lesions were located in the buccal mucosa, tongue, and gingiva. Among patients with leukoplakia, smoking was the most common habit, followed by tobacco chewing, whereas tobacco chewing was predominant among patients with OSCC, followed by gutka use. Following qPCR quality control, two healthy control samples were excluded because of undetectable target or reference gene amplification, resulting in a final analytical cohort of 43 participants (13 healthy controls, 15 with leukoplakia, and 15 with OSCC).

Fig. 1: Clinical photographs of healthy controls (A), patients with oral leukoplakia (B), and oral squamous cell carcinoma (C).

Salivary Telomere length analysis

The individual raw ΔΔCt values were converted to Relative Telomere Length (RTL) ratios using the formula 2−ΔΔCt. Because RTL values were markedly right-skewed and failed the Shapiro-Wilk test of normality in every group (p < 0.001), the median and IQR are reported as the primary summary statistics, with the mean ± SD given for reference. The median RTL value for the healthy control group (n = 13) was 0.0116 (IQR 0.0015–0.1393; mean ± SD 0.4055 ± 1.0020). For the oral leukoplakia cohort (n = 15), the median RTL was 0.0604 (IQR 0.0269–0.4296; mean ± SD,0.4751 ± 0.7967). The oral squamous cell carcinoma (OSCC) cohort (n = 15) exhibited a median RTL of 0.0516 (IQR 0.0230–1.0507; mean ± SD 0.6060 ± 0.8730) (Fig. 2).

Fig. 2: Distribution of salivary relative telomere length (RTL) among healthy controls (n = 13), oral leukoplakia (n = 15), and oral squamous cell carcinoma (OSCC) (n = 15). Box-and-whisker plots represent the median (central line), interquartile range (box), and whiskers representing the range of non-outlier observations, with individual participant values overlaid. Considerable overlap and inter-individual variability were observed across the three groups, consistent with the absence of a statistically significant difference in RTL (Kruskal–Wallis test, p = 0.261).

Because RTL values were markedly right-skewed and failed the Shapiro-Wilk test of normality in every group (p < 0.001), the non-parametric Kruskal-Wallis test was used instead of one-way ANOVA (Table 2). This test showed no statistically significant difference in RTL among the three groups (H (2) = 2.68, p = 0.261). Post-hoc pairwise comparisons using the Mann-Whitney U test with Bonferroni correction likewise showed no statistically significant differences between any two groups (normal vs. leukoplakia, p = 0.748; leukoplakia vs. OSCC, p = 1.000; normal vs. OSCC, p = 0.352; Table 3).

Diagnostic Group | Sample Size (n) | Mean RTL (2−ΔΔCt​) | Median RTL (IQR) | Standard Deviation (SD) | Global Statistical Significance
Healthy Controls | 13 | 0.4055 | 0.0116 (0.0015–0.1393) | 1.0020 | Shapiro-Wilk: p < 0.001 (non-normal); Levene's p = 0.850
Oral Leukoplakia | 15 | 0.4751 | 0.0604 (0.0269–0.4296) | 0.7967 | Kruskal-Wallis:H(2) = 2.68, p = 0.261
OSCC | 15 | 0.6060 | 0.0516 (0.0230–1.0507) | 0.8730 | 

Comparison | Mann–Whitney U | p (uncorrected) | p (Bonferroni) | r value
Normal vs Leukoplakia | 123.0 | 0.249 | 0.748 (NS) | −0.26
Leukoplakia vs OSCC | 103.0 | 0.709 | 1.000 (NS) | 0.08
Normal vs OSCC | 63.0 | 0.117 | 0.352 | 0.35