Title: A standalone approach to utilize telomere length measurement as a surveillance tool in oral leukoplakia

Oral Squamous Cell Carcinoma (OSCC) is often preceded by white patch, called oral leukoplakia (OL). Assessing relative telomere length (TL) in OL could be a predicting biomarker. Due to high variability and lack of universal reference, there has been a limited translational application of TL. Here, we describe an approach of evaluating TL using paired PBMC as internal reference and demonstrate its translational relevance. Oral brush biopsy and paired venous blood were collected from 50male OL patients and 44male healthy controls. Relative TL was measured by qPCR. TL of each OL sample was normalized to paired PBMC sample (TL ratio). Mean TL ratio in healthy controls with high risk oral habits, was shorter than those who did not have these habits (1.093 {+/-} 0.411 and 1.253 {+/-} 0.296, respectively; p=0.071). In OL patients, the mean TL ratio was not only significantly shorter in the patch but also in distal normal oral tissue (0.971 {+/-} 0.317, p=0.0002 and0.896 {+/-} 0.284, p=0.00001, respectively), relative to healthy control without high risk oral habit. Based on the TL ratio, we proposed a classification of OL into four subgroups. Dysplastic pathology was frequently associated with a subgroup having normal TL ratio at patch while significantly shorter TL ratio at paired normal distal site. The approach of analyzing TL attrition of oral mucosa, eliminating requirement of external reference DNA, will enable the TL data universally comparable and provide a useful marker to define high risk OL group for follow-up program. Larger studies will further validate the approach and its broader application in other pre-malignant conditions.