Abstract
BackgroundOral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need.Methodology/Principal FindingsTo discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells.Conclusions/SignificanceSalivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early.
Highlights
Oral cancer develops in stages, transitioning from a normal oral epithelium, to a pre-malignant, dysplastic oral lesion, to a malignant lesion, most commonly in the form of oral squamous cell carcinoma (OSCC)
Clinical whole saliva collection protocol from subjects Unstimulated whole saliva was collected from two groups: subjects with pre-malignant dysplastic lesions diagnosed by scalpel biopsy and histology, and subjects with invasive malignant lesions, diagnosed as primary OSCC by scalpel biopsy and histology
We have described in detail the procedure used for pulsed Q dissociation (PQD) analysis of iTRAQ reagent labeled peptides previously, as well as the HPLC and LTQ operating conditions for tandem mass spectrometry (MS/MS) analysis[14]
Summary
Oral cancer develops in stages, transitioning from a normal oral epithelium, to a pre-malignant, dysplastic oral lesion, to a malignant lesion, most commonly in the form of oral squamous cell carcinoma (OSCC) For those who develop OSCC, the overall 5- year survival rate is approximately 50%, unchanged over the last 30 years[1]. The current gold standard for characterizing lesions, histological analysis of tissue biopsies[3], has several disadvantages: expert clinicians are required to collect the samples and interpret results, incurring relatively high costs; inaccuracies in diagnosis due to difficulties sampling tissue which may have multiple dysplastic foci; and patient discomfort with the procedure Together these disadvantages limit the accuracy of diagnosis, the frequency of patient testing, and the ability to detect oral cancer early by tissue biopsy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need
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