Oral squamous cell carcinoma (OSCC) accounts for >90% of cases of oral cancer, including cancer at the lip and oral cavity and cancer at the oropharynx. Most OSCCs develop from oral potentially malignant disorders (OPMDs), which consist of heterogeneous lesions with different malignant transformation potentials that make early detection of OSCC a challenge. Using a targeted mass spectrometry-based assay to compare multiple candidate proteins, we previously identified matrix metalloproteinase-1 (MMP-1) as one of the most promising salivary OSCC biomarkers. To explore the clinical utility of MMP-1 in OSCC detection, we developed an in-house, sensitive enzyme-linked immunosorbent assay (ELISA) for measuring MMP-1 content, and tested it on saliva samples from 1160 subjects (313 healthy controls, and 578 OPMD and 269 OSCC patients) collected at two medical centers. Salivary MMP-1 levels measured by our in-house ELISA significantly discriminated OSCC patients from non-cancerous groups. A receiver operating characteristic curve analysis showed that MMP-1 was effective in separating non-cancer groups from patients with OSCCs at the oral cavity. Additionally, salivary MMP-1 levels in oral cavity cancer patients were highly correlated with tumor progression (tumor size, lymph node metastasis, and overall stage). Collectively, our results indicate that salivary MMP-1 is an effective biomarker for OSCC that can be sensitively detected using our newly developed ELISA. The newly developed MMP-1 ELISA may be used as a new adjunctive tool to aid in detecting and monitoring OSCC.

Extracellular vesicles (EVs) are valuable sources for the discovery of useful cancer biomarkers. This study explores the potential usefulness of tumor cell-derived EV membrane proteins as plasma biomarkers for early detection of colorectal cancer (CRC). EVs were isolated from the culture supernatants of four CRC cell lines by ultracentrifugation, and their protein profiles were analyzed by LC-MS/MS. Bioinformatics analysis of identified proteins revealed 518 EV membrane proteins in common among at least three CRC cell lines. We next used accurate inclusion mass screening (AIMS) in parallel with iTRAQ-based quantitative proteomic analysis to highlight candidate proteins and validated their presence in pooled plasma-generated EVs from 30 healthy controls and 30 CRC patients. From these, we chose 14 potential EV-derived targets for further quantification by targeted MS assay in a separate individual cohort comprising of 73 CRC and 80 healthy subjects. Quantitative analyses revealed significant increases in ADAM10, CD59 and TSPAN9 levels (2.19- to 5.26-fold, p < 0.0001) in plasma EVs from CRC patients, with AUC values of 0.83, 0.95 and 0.87, respectively. Higher EV CD59 levels were significantly correlated with distant metastasis (p = 0.0475), and higher EV TSPAN9 levels were significantly associated with lymph node metastasis (p = 0.0011), distant metastasis at diagnosis (p = 0.0104) and higher TNM stage (p = 0.0065). A two-marker panel consisting of CD59 and TSPAN9 outperformed the conventional marker CEA in discriminating CRC and stage I/II CRC patients from healthy controls, with AUC values of 0.98 and 0.99, respectively. Our results identify EV membrane proteins in common among CRC cell lines and altered plasma EV protein profiles in CRC patients and suggest plasma EV CD59 and TSPAN9 as a novel biomarker panel for detecting early-stage CRC.

Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.