Salivary proteomics for oral cancer biomarker discovery

Abstract

Purpose: This study aims to explore the presence of informative protein biomarkers in the human saliva proteome and to evaluate their potential for detection of oral squamous cell carcinoma (OSCC).

Experimental design: Whole saliva samples were collected from patients (n = 64) with OSCC and matched healthy subjects (n = 64). The proteins in pooled whole saliva samples of patients with OSCC (n = 16) and matched healthy subjects (n = 16) were profiled using shotgun proteomics based on C4 reversed-phase liquid chromatography for prefractionation, capillary reversed-phase liquid chromatography with quadruple time-of-flight mass spectrometry, and Mascot sequence database searching. Immunoassays were used for validation of the candidate biomarkers on a new group of OSCC (n = 48) and matched healthy subjects (n = 48). Receiver operating characteristic analysis was exploited to evaluate the diagnostic value of discovered candidate biomarkers for OSCC.

Results: Subtractive proteomics revealed several salivary proteins at differential levels between the OSCC patients and matched control subjects. Five candidate biomarkers were successfully validated using immunoassays on an independent set of OSCC patients and matched healthy subjects. The combination of these candidate biomarkers yielded a receiver operating characteristic value of 93%, sensitivity of 90%, and specificity of 83% in detecting OSCC.

Conclusion: Patient-based saliva proteomics is a promising approach to searching for OSCC biomarkers. The discovery of these new targets may lead to a simple clinical tool for the noninvasive diagnosis of oral cancer. Long-term longitudinal studies with large populations of individuals with oral cancer and those who are at high risk of developing oral cancer are needed to validate these potential biomarkers.

Fig. 1

Subtractive proteomics and 2-DE profiling of proteins in pooled WS samples from 16 OSCC or 16 matched control subjects. A, the intact proteins in each pooled sample were initially prefractionated using C4 reversed-phase LC. In total, 35 fractions were collected from each pooled sample and proteins in each fraction were reduced, alkylated, and digested using trypsin. The resulting peptides were analyzed using capillary LC-QqTOF MS/MS. B, database searching using Mascot was carried out to identify a total of 461proteins from OSCC subjects and 438 proteins from the matched control subjects. Most proteins (n = 409) overlap between the disease and control samples. However, 52 proteins were only found in OSCC, whereas 29 proteins were only identified in the healthy subjects. C, 2-DE patterns of WS proteins from the same pooled cancer and control subjects. The circled spots were identified to be PIGR, which is a down-regulated glycoprotein in OSCC.

Fig. 2

A, validation of five candidate biomarkers (M2BP, MRP14, CD59, catalase, and profilin) between OSCC (n = 48) and matched control (n = 48) subjects. MRP14, CD59, catalase, and profilin were measured by Western blotting and normalized against corresponding actin levels, whereas M2BP was measured with ELISA (μg/mL). B, ROC analysis based on the validation results of these five candidate markers. The area under the curve (AUC) was determined as 0.93. The sensitivity and specificity were 90% and 83%, respectively.