Purpose: Oral fluid (saliva) meets the demand for noninvasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical approach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease.Experimental Design: Unstimulated saliva was collected from patients (n ‫؍‬ 32) with primary T1/T2 OSCC and normal subjects (n ‫؍‬ 32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models.Results: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05).Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n ‫؍‬ 32) and controls (n ‫؍‬ 32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combinations of these biomarkers yielded sensitivity (91%) and specificity (91%) in distinguishing OSCC from the controls.Conclusions: The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be exploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveillance.

Objective The associations between oral diseases and increased risk of pancreatic cancer have been reported in several prospective cohort studies. In this study, we measured variations of salivary microbiota and evaluated their potential associations with pancreatic cancer and chronic pancreatitis. Methods This study was divided into three phases: (1) microbial profiling using the Human Oral Microbe Identification Microarray to investigate salivary microbiota variation between 10 resectable patients with pancreatic cancer and 10 matched healthy controls, (2) identification and verification of bacterial candidates by real-time quantitative PCR (qPCR) and (3) validation of bacterial candidates by qPCR on an independent cohort of 28 resectable pancreatic cancer, 28 matched healthy control and 27 chronic pancreatitis samples. Results Comprehensive comparison of the salivary microbiota between patients with pancreatic cancer and healthy control subjects revealed a significant variation of salivary microflora. Thirty-one bacterial species/clusters were increased in the saliva of patients with pancreatic cancer (n=10) in comparison to those of the healthy controls (n=10), whereas 25 bacterial species/clusters were decreased. Two out of six bacterial candidates (Neisseria elongata and Streptococcus mitis) were validated using the independent samples, showing significant variation (p<0.05, qPCR) between patients with pancreatic cancer and controls (n=56). Additionally, two bacteria (Granulicatella adiacens and S mitis) showed significant variation (p<0.05, qPCR) between chronic pancreatitis samples and controls (n=55). The combination of two bacterial biomarkers (N elongata and S mitis) yielded a receiver operating characteristic plot area under the curve value of 0.90 (95% CI 0.78 to 0.96, p<0.0001) with a 96.4% sensitivity and 82.1% specificity in distinguishing patients with pancreatic cancer from healthy subjects. Conclusions The authors observed associations between variations of patients’ salivary microbiota with pancreatic cancer and chronic pancreatitis. This report also provides proof of salivary microbiota as an informative source for discovering non-invasive biomarkers of systemic diseases.

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.

Objective. To identify a panel of protein and messenger RNA (mRNA) biomarkers in human whole saliva (WS) that may be used in the detection of primary Sjögren's syndrome (SS).Methods. Mass spectrometry and expression microarray profiling were used to identify candidate protein and mRNA biomarkers of primary SS in WS samples. Validation of the discovered mRNA and protein biomarkers was also demonstrated using real-time quantitative polymerase chain reaction and immunoblotting techniques.Results. Sixteen WS proteins were found to be down-regulated and 25 WS proteins were found to be up-regulated in primary SS patients compared with matched healthy control subjects. These proteins reflected the damage of glandular cells and inflammation of the oral cavity system in patients with primary SS. In addition, 16 WS peptides (10 up-regulated and 6 downregulated in primary SS) were found at significantly different levels (P < 0.05) in primary SS patients and controls. Using stringent criteria (3-fold change; P < 0.0005), 27 mRNA in saliva samples were found to be significantly up-regulated in the primary SS patients. Strikingly, 19 of 27 genes that were found to be overexpressed were interferon-inducible or were related to lymphocyte filtration and antigen presentation known to be involved in the pathogenesis of primary SS.Conclusion. Our preliminary study has indicated that WS from patients with primary SS contains molecular signatures that reflect damaged glandular cells and an activated immune response in this autoimmune disease. These candidate proteomic and genomic biomarkers may improve the clinical detection of primary SS once they have been further validated. We also found that WS contains more informative proteins, peptides, and mRNA, as compared with gland-specific saliva, that can be used in generating candidate biomarkers for the detection of primary SS.

Purpose-Detection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome.Patients and Methods-qRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (β1→4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3).Results-Fifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P < .0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of ≥ one marker in histopathologynegative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P = .0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1).Conclusion-Molecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.