Will oral health providers one day be able to scout for evidence of systemic disease in the time it takes to clean a patient’s teeth? It’s an ever-likely scenario, according to those at the forefront of salivary diagnostics research.
Clinical discoveries published in recent years have advanced the odds of salivary diagnostics becoming a chairside tool that could enhance the ability of dental practitioners to detect a spectrum of medical conditions.
Scientists working with saliva samples obtained from a group of Indian patients, for example, were able to validate the ability of portable electromechanical biosensor hardware technology to simultaneously analyze oral fluids for biomarkers thought to signal the pathogenesis of oral cancer (Clinical Cancer Research, July 1, 2009, Vol. 15:13, pp. 4446-4452).
A similar device developed by scientists and engineers in California sits at the cusp of commercialization (Annals of the New York Academy of Science, March 2007, Vol. 1098, pp. 401-410). The prototype Oral Fluid Nanosensor Test (OFNASET) awaits regulatory approval, with two contract manufacturing organizations waiting in the wings to begin commercialization.
And numerous studies funded by the National Institutes of Health (NIH) and others in 2009 and 2010 have pushed the salivary diagnostics envelope to its most advanced position yet, with molecular biomarkers being identified to monitor the systemic creep of breast, ovarian, pancreatic, and lung cancers.
And these advances represent just a portion of the salivary diagnostics portfolio. Combinations of analytes found in whole and ductal saliva are being characterized in ways that allow investigators to view them as molecular "proxies" for a host of systemic or systemic-related conditions, including periodontal infection, diabetes, human immunodeficiency virus (HIV), hepatitis, and cardiovascular disease.
In addition, investigators continue to refine ways to link the basic investigative tools of molecular biology — such as mass spectrometry and microarray analysis — with microfluidics and nanotechnology engineering, enabling them to develop portable salivary assay platforms for use in the dental operatory. If myriad challenges associated with regulatory approval, licensing, commercialization, and third-party reimbursement can be met, oral care providers may one day soon find themselves offering diagnostic health services that can identify life-threatening conditions.
Painless chairside option
The idea of an inexpensive and painless chairside option to blood or urine collection for disease diagnosis captivates the imagination of people like David Wong, DMD, DMSc, the associate dean of research at the University of California, Los Angles (UCLA) and director of the Dental Research Institute at the UCLA School of Dentistry.
"There’s something very magical about salivary diagnostics," he said in an interview with DrBicuspid.com. "It has this resonance. It just echoes within the profession. Seemingly, there’s this yearning for the profession to be able to move itself forward through a scientifically credible portfolio that, at least in the very beginning, they can claim is theirs."
Dr. Wong’s name is attached to scores of papers on the subject. He and others have published prolifically on salivary diagnostics since 2002, when the National Institute of Dental and Craniofacial Research (NIDCR) awarded seven NIH grants to pursue development of a microelectromechanical systems approach to the nascent discipline. Funding was extended to a consortium of researchers that included Dr. Wong’s group and scientists at the Scripps Research Institute, the University of Rochester, the University of Southern California, and the University of California, San Francisco.
The Wong Lab has since earned a special place in the research spotlight for its oncology studies, its collaborative work on the OFNASET portable device, and its sponsorship of the Salivaomics Knowledge Base (SKB). The latter is a collection of data "alphabets" describing the accumulated knowledge of saliva’s molecular constituents, with primary emphasis on proteins and messenger RNA (mRNA) transcripts and secondary attention devoted to the metabolites, micro-RNAs, and microbes in oral fluids.
Dr. Wong acknowledged that the clinical utility of protein- and mRNA-specific alphabets in the SKB has so far eclipsed that of the other three analyte categories.
"Based on our own experience, what is important is to have the best capability to find the most discriminatory markers," he explained. "Using all five [alphabets] in any disease is ideal, but it’s not practical."
The discovery of biomarkers via two-dimensional gel electrophoresis, for proteins, and reverse transcription polymerase chain reaction methodology, used to trace mRNA transcripts back to their complementary DNA, represents just two of the numerous methods being deployed to complete a portion of the salivary diagnostics puzzle. For their part, engineers are challenged to wed technologies that yield guidance at the proteome- and genome-wide level with compact nano- or micro-scaled platforms that can, at chairside, winnow the evidence down further to highly discriminatory panels of disease-specific biomarkers.
Microfluidics systems might be rendered in various combinations that merge components such as sensors or actuators with silicon substrates, allowing for the site-specific analysis of saliva samples and reagents. In the 2009 Clinical Cancer Research study of saliva from Indian patients, for example, a portable system was able to quantify interleukin biomarkers with a sensitivity and specificity nearly equal to that of traditional assay techniques. These claims are also made for the OFNASET device.
Oral cancer and Sjögren’s syndrome
As new tools and assays emerge, investigators are expanding and refocusing the theoretic boundaries of oral fluid diagnostics while maintaining an investigative focus on those diseases that hold the greatest promise for application of the science in the near- to midterm.
"As I scan around, what I see that’s in the pipeline is oral cancer and Sjögren’s syndrome," Dr. Wong said. "That’s what I’m seeing. There are other developments as well, but they’re very, very early."
Oral squamous cell carcinoma (OSCC) and Sjögren’s syndrome have indeed commanded a great deal of attention under NIDCR’s proof-of-concept umbrella. A 2007 study in Arthritis & Rheumatism (November 2007, Vol. 56:11, pp. 3588-3600) found that 16 whole saliva proteins were down-regulated and 25 more were up-regulated in patients with primary Sjögren’s syndrome, an autoimmune disorder characterized by dry mouth and eyes. These variations in expression were acknowledged as a reflection of glandular cell damage and an activated immune response.
Major findings specific to OSCC since 2004 have included explorations of mRNA transcriptome factors, the identification of three tumor markers at elevated levels in saliva (Cyfra 21-1, tissue polypetide antigen, and CA125), and the description of five salivary proteins (M2BP, MRP14, profilin, CD59, and catalese) that were shown to discriminate the presence of OSCC with 90% sensitivity and 83% specificity (Clinical Cancer Research, December 15, 2004, Vol. 10:24, pp. 8442-8450; July 1, 2006, Vol. 12:13, pp. 3979-3984; October 1, 2008, Vol. 14:19, pp. 6246-6252).
Elucidation of the biomarkers for these two diseases sets the stage for further studies to refine and validate them. Trial results also encouraged additional efforts aimed at ferreting out the salivary pointers to other malignancies. Evidence published in 2010 described protein and mRNA biomarkers for breast and pancreatic cancers (PLoS One, December 31, 2010, Vol. 5:12, e15573; Gastroenterology, March 2010, Vol. 138:3, pp. 949-957.e7). More recently, salivary biomarkers have been isolated for ovarian and lung cancers (Journal of Molecular Medicine, November 18, 2011; Molecular & Cellular Proteomics, November 17, 2011). These newest studies report biomarker sensitivities ranging between 83% and 96.4% and disease specificity measures ranging from 91.4% to 97%.
Early research critical
So why wasn’t a fluid as ubiquitous as spit considered an effective diagnostic medium before? Saliva, after all, is a filtrate of the blood, and a liter or more of it issues each day from three major oral glands and a multitude of minor glands. Our mouths leverage the digestive function, facilitate our sense of taste, regulate pH balances, and bathe the oral cavity in a soup of minerals, antibacterials, enzymes, antibodies, hormones, electrolytes, and growth factors.
Dental practitioners, however, have regarded saliva more as a helpful nuisance than a harbinger of ill health, in large part because the telltale analytes associated with major categories of disease are much more concentrated in blood and urine than they are in oral fluids.
Efforts to get saliva off the bench and onto the diagnostic playing field got a major boost in 2003 and 2004 when various researchers collaborated under NIH-NIDCR sponsorship to document the salivary proteome. Eventually, 1,166 proteins were identified, 657 of which are also present in blood plasma. Researchers found that extracellular proteins excreted into the spaces between cells were more abundant in saliva than in plasma, while proteins located in or near lipid membranes were seen more frequently in plasma than in saliva.
Additional research published in 2004 found that saliva contains more than 3,000 chemically distinct mRNAs. A few of these were observed to comprise a core signature of mRNAs that typically are found in healthy individuals.
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