Oral Squamous Cell Carcinoma (OSCC) is one of the most prevalent cancers in the world with the maximum number of cases reported from India. Poor survival rate associated with OSCC can be attributed to the non-availability of a biomarker, as one of the major reasons, leading to late presentation. Identification of an early diagnostic biomarker, which can also be used as a screening tool, will be helpful in reducing the disease morbidity and mortality. In this report, we present Parallel Reaction Monitoring (PRM) based validation of 12 candidate proteins, identified initially by tandem mass tag (TMT) based relative quantification of salivary proteins on LC-MS, in the saliva of Oral Squamous Cell Carcinoma (OSCC) cases (N=50) and healthy controls (N=49), AZGP1, AHSG, KRT6C, S100A7, S100A9, KLK1, BPIFB2, IGLL5, CORO1A, LACRT, LCN2 and PSAP. Heavy isotope labelled reference peptides were used to produce calibration curve and absolute quantification of proteins and resulting data were analyzed statistically using R. Salivary AHSG (p=0.0041**) and KRT6C (p=0.002**) were significantly upregulated in OSCC cases while AZGP1 (p<0.0001***), KLK1 (p=0.006**) and BPIFB2 (p=0.0061**) were significantly downregulated. Multivariate logistic regression modelling resulted in a better risk prediction model consisting of AZGP1, AHSG and KRT6C with p value <0.0001***. Using this model ROC with an area under the curve of 82.2% was produced and sensitivity and specificity observed for this model was 78% and 73.5%, higher than any of the individual proteins. Positive and negative predictive values for the model were 76% and 75% respectively. We report a potential biomarker panel consisting of proteins AZGP1, AHSG and KRT6C for early diagnosis of OSCC.

A Parallel Reaction Monitoring method was developed to estimate the levels of salivary peptides in the saliva samples of OSCC cases and healthy controls. For absolute quantification, 10 different standard concentrations were acquired.

A prospective case-control study was designed. Patients attending the Department of Radiotherapy and Department of Otolaryngology at Post Graduate Institute of Medical Education and Research, Chandigarh (India), undergoing surgery and/or receiving the standard radio/chemo-therapy with curative intent based on disease stage, decided as per the approved clinical protocol in the institute were enrolled in the study. Fifty, biopsy-proven OSCC cases and age and gender matched 49 healthy volunteers were recruited after obtaining informed consent form and following the inclusion and exclusion criteria (supplementary data). Unstimulated saliva samples (at least 5 ml of saliva) were collected, following at least half an hour abstinence from any food and fluid including water, by collecting the saliva directly in a 50mL centrifuge tube. The collected sample was centrifuged at 5000 rpm for 20 minutes at 4°C and supernatant was collected and preserved at -80°C for further analysis.