Discussion

Telomeres are repetitive DNA sequences located at the chromosomal termini that maintain genomic stability. Progressive telomere shortening during successive cell divisions normally triggers cellular senescence or apoptosis, whereas dysregulation of telomere maintenance through telomerase reactivation or the alternative lengthening of telomeres (ALT) pathway allows cells to bypass this checkpoint and contributes to genomic instability and tumorigenesis.16,17

The literature on telomere dynamics in oral carcinogenesis is notably not unanimous in its direction. A substantial body of work, including Sainger et al.,18 Aida et al.'s tissue and Q-FISH studies, and Zhu et al.'s population-level meta-analysis,19,20 reports that oral precancerous and cancerous tissues tend to show shortened telomeres relative to normal mucosa, consistent with the classical model in which attrition precedes and permits malignant transformation. In contrast, there is a separate line of evidence that is largely focused on telomerase activity and expression rather than length per se, reporting a progressive increase across the normal-to-precancer-to-cancer sequence. Samadi et al. found significantly higher telomerase activity in OSCC than in precancerous lesions and proposed that augmented telomerase expression, together with telomere length changes, contributes to OSCC progression and could serve as an early diagnostic biomarker.21 This is corroborated by immunohistochemical data showing that the hTERT labelling index and labelling score increase stepwise from normal oral mucosa through leukoplakia to OSCC,22 and by Rai et al.'s finding of elevated telomerase activity in OSCC relative to normal oral mucosal tissue.23 Pannone et al. similarly reported increased hTERT gene expression across the oral carcinogenesis continuum.24

In the present study, salivary telomere length was evaluated in patients with leukoplakia and OSCC relative to healthy controls. Contrary to our initial hypothesis of a linear reduction in telomere length during progressive transformation, our data revealed a highly variable, statistically non-significant upward trend in relative telomere length ratios (2−ΔΔCt) from healthy controls (0.41) to leukoplakia (0.48), and OSCC (0.61). Bonferroni-corrected post hoc pairwise comparisons (Mann–Whitney U) likewise showed no significant differences between any two groups (normal vs. leukoplakia, p = 0.748; leukoplakia vs. OSCC, p = 1.000; normal vs. OSCC, p = 0.352), reinforcing that the apparent upward trend does not reach significance at any stage of transformation. One possible explanation for this observation is the reactivation of telomerase or the engagement of alternative lengthening of telomeres (ALT) mechanisms during oral carcinogenesis. These mechanisms may contribute to the heterogeneous telomere length measurements in saliva. However, as telomerase activity and ALT were not evaluated in the present study, this explanation remains speculative and requires further investigation. Pal et al. observed a related phenomenon directly in oral leukoplakia, where telomeres in the lesion patch were somewhat longer and less consistent than those in paired normal oral mucosa, which they attributed to the localized reactivation of telomere maintenance mechanisms within the lesion.25 A similar mechanism may explain the variability observed in the present study.

The wide intra-group variation and higher mean RTL observed in the leukoplakia and OSCC groups may be explained by several factors. First, telomerase or ALT-pathway upregulation is a recognized hallmark of transformed and pre-transformed oral epithelial cells seeking to evade apoptosis, and can produce highly elongated or heterogeneous telomere distributions even within a single lesion.18 Second, whole saliva is a complex bioliquid containing not only exfoliated epithelial cells but also a substantial and variable influx of local inflammatory leukocytes and exudate, particularly under the chronic inflammatory conditions that accompany leukoplakia and OSCC. Because immune cells maintain comparatively long telomeres, their variable presence in mixed salivary DNA can mask epithelial-specific shortening and generate the kind of noisy, upward-skewed signal we observed.26

Additional contributors to telomere alteration likely include oxidative stress, chronic inflammation, and increased cellular turnover,26 with tobacco and betel quid use, the predominant habits in our leukoplakia and OSCC cohorts, further amplifying this oxidative damage.27 The utility of saliva as a diagnostic medium is supported by evidence that salivary telomere length correlates with peripheral blood telomere dynamics,28 reinforcing its plausibility as a non-invasive proxy for systemic and local telomere biology, even when the present whole-saliva measurements proved too heterogeneous to reach significance.

This study demonstrated a non-significant upward trend in relative salivary telomere length in oral leukoplakia and OSCC. Although this pattern is consistent with previous studies reporting increased telomerase activity during oral carcinogenesis, the present study did not assess telomerase activity or hTERT expression levels. Therefore, no conclusions regarding the underlying biological mechanisms can be drawn from these studies.

Limitations. The present study has limitations, including the relatively small sample size, cross-sectional design, absence of technical qPCR replicates and amplification efficiency analysis, and the heterogeneous cellular composition of whole saliva, which may have influenced the relative telomere length measurements. In addition, potential confounding factors, such as tobacco habits, age, and sex, were not adjusted for in the multivariable analyses. The absence of technical replicates, necessitated by the limited genomic DNA yield from low-volume saliva collections, is a further limitation of this exploratory pilot study. This was partially mitigated by running all target and reference reactions on a single 96-well plate to eliminate inter-plate variation, together with strict melt curve validation of every well. Consequently, the observed relative telomere length measurements should be regarded as preliminary and require validation in future studies that incorporate technical replicates and larger independent cohorts. Larger longitudinal studies incorporating technical replicates and cellular fractionation of salivary DNA paired with direct telomerase activity or hTERT expression assays are needed.

Conclusion: This preliminary case–control study evaluated the potential of salivary relative telomere length as a non-invasive biomarker for oral carcinogenesis. Although a non-significant increasing trend was observed from healthy controls to oral leukoplakia and OSCC, the substantial inter-individual variability and lack of statistical significance suggest that whole saliva relative telomere length is unlikely to serve as a reliable standalone biomarker in its current form. Nevertheless, these findings provide preliminary insights into telomere biology in oral carcinogenesis and warrant further validation in larger longitudinal studies incorporating technical replicates, cellular fractionation, and complementary assessments of telomerase activity or hTERT expression.