Quantitative Proteomic Analysis Reveals Decreased Salivary Amylase in Oral Cancer

Objectives: Our lab is engaged in identification of novel biomarkers which may predict transformation of oral leukoplakia (OL) to squamous cell carcinoma( SCC) using a discovery-based proteomic approach to determine the relative abundance of proteins in saliva.  Methods: Saliva specimens were collected from 14 OL and SCC patients diagnosed by clinical, histological and serological as well as control age-gender-ethnicity matched healthy individuals. Samples were centrifuged after collection to remove exfoliated cells and supernatants were pooled under each group. Protein amounts were determined using the BCA protein assay. Equal amounts (60 µg) of each pooled sample were denatured and reduced prior to trypsin digestion.  All peptides in each pooled sample were then labeled with an amine specific iTRAQ reporter that binds to lysine and amino termini. After combining all three pooled samples and drying in a SpeedVac, a Sep-Pak C18 cartridge was used. The recovered peptide mixture was analyzed by 3-D fractionation and tandem mass spectrometry. The data were searched using Sequest 27.0 and quantitation performed by in-house software. Results: Amylase abundance and activity was significantly reduced in the SCC patients vs. the OL and control patients (p<0.05 ) . Additionally, there was a difference in amylase abundance and activity between smokers and non-smokers. Conclusions: This pilot study demonstrates that the abundance and activity of salivary amylase distinguishes OPML from OSCC; At least one other group has reported that amylase abundance may drop in oral cancer patients (Shpitzer, T., et al.); factors other than OSCC that may account for these observations were tested and did not show significant effects; patients with metastatic breast cancer did not show similar amylase results (not a general effect of metastasis). These overall results point to possibility of using amylase activity to monitor patients with OPML which may indicate transformation to OSCC.