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Imagine the benefits of being able to monitor health status, disease onset and progression, and treatment outcomes through noninvasive means. Salivary diagnostics could turn this possibility into a reality. A national initiative promoted by the National Institute of Dental and Craniofacial Research (NIDCR) has created a roadmap to turn saliva diagnostics into a clinical reality. Oral fluid is a perfect medium in the exploration of health and in disease surveillance. The clinical applications and opportunities are enormous. 

 

 
Figure 12
Anatomical locations of the three major salivary glands: parotid, submandibular, and sublingual.

By permission of Mayo Foundation for Medical Education and Research. All rights reserved.
 
Human diseases, such as cancer and cardiovascular, metabolic, and neurological diseases, are challenging to diagnose without laboratory testing in addition to clinical evaluation. Even with laboratory tools, definitive diagnosis often remains elusive. Saliva, a bodily fluid that can be obtained noninvasively and is readily available, has long been recognized as harboring such diagnostic potential.1 With the NIDCR’s support, saliva biomarker discovery and salivary diagnostic technologies are currently in active development. The use of saliva for disease diagnostics and normal health surveillance is most likely about 5 years away. This will truly allow the bridging of oral health research into systemic diseases through the biofluid that filters and processes itself from the vasculature that nourishes the salivary glands (Figures 1 and 2). Understanding that disease markers pass from blood into saliva provides the opportunity to use saliva for diagnostic applications to systemic diseases.
 
Figure 2 2
Mechanisms of transport of proteins and ions from serum into salivary gland ducts.

By permission of Mayo Foundation for Medical Education and Research. All rights reserved.
 

The pool of evidence is growing to support the use of saliva in monitoring systemic diseases and conditions. The barriers to widespread implementation of salivary diagnostics have been largely overcome. Techniques are emerging from a combination of miniaturization technologies and discoveries in many different fields including biology, chemistry, physics, and engineering that are leading to high throughput (the ability to process and analyze a large number of samples at the same time), automated, portable, low cost, more efficient, and rapid biochemical analyses. Miniaturized diagnostic technologies will be able to yield patient information reflecting health and disease status with minute amounts of body fluids. These lab-on-a-chip platforms will be able to perform multiple operations in parallel in nonlaboratory settings such as the field, factory, hospital clinic, or home. Such technologies will allow the simultaneous assessment of multiple conditions of health and disease and provide clinicians with prevention and therapeutic strategies to meet patient needs. 

VISION AND CHALLENGES 

The vision of salivary diagnostics is to use the diagnostic potential and ­optimize engineering tech­nologies for this body fluid. Figure 3 is a ven diagram that illustrates that within the spectrum of total human health and disease states (top circle), some will reflect themselves diagnostically in saliva through proteomic or genomic information (lower left circle). How much overlap the subsets have remains to be seen. The lower right circle illustrates the technology development platforms necessary to advance the detection capability of saliva. 

The challenge to make salivary diagnostics a clinical reality is in establishing the scientific foundation and clinical validations necessary to position it as a highly accurate and feasible technology that can achieve definitive point-of-care assessment of patients’ health and disease status. Inherent in this vision is the attainment of the science and diagnostic targets in saliva and the development of robust, simple-to-use, biosensor technologies for reliable and valid clinical applications. 

SALIVA AS A DIAGNOSTIC FLUID 

Saliva is the body’s mirror. The ability to use saliva to monitor the health and disease state of an individual is a highly desirable goal for health promotion and health care research. However, the growing appreciation of saliva as a mirror that can reflect virtually the entire spectrum of normal and disease states is relatively recent.1 Saliva can reflect tissue levels of natural substances and a large variety of molecules introduced for therapeutic, dependency, or recreational purposes; emotional status; hormonal status; immunological status; neurological effects; and nutritional and metabolic influences. A major drawback to using saliva as a diagnostic fluid is the fact that informative analytes (substances undergoing analysis) are generally present in lower amounts in saliva than in blood.3 However, with new and highly sensitive techniques, the lower level of analytes in saliva is no longer a limitation. Almost anything that can be measured in blood, can be measured in saliva. Saliva has been reliably used to detect HIV 1 and 2 and viral hepatitis A, B, and C. It can also be used to monitor a variety of drugs including marijuana, cocaine, and alcohol.1 

 
Figure 3
Disease markers manifestation in saliva and their detection by salivary diagnostic biosensors (Oral Fluid NanoSensor Test, OFNASET).
 

Saliva is effective as a diagnostic fluid to monitor health and diseases as it is inexpensive, noninvasive, and easy-to-use. As a clinical tool, saliva has many advantages over blood. Saliva is easy to collect, store, and ship, and it can be obtained at low cost and in sufficient quantities for analysis. For patients, the noninvasive collecting techniques dramatically reduce anxiety and discomfort and simplify the gathering of repeated samples for longitudinal monitoring over time. For professionals, saliva collection is safer than blood tests, which can expose health care providers to HIV or hepatitis virus. Saliva is also easier to handle for diagnostic procedures since it does not clot, lessening the manipulations required. Saliva-based diagnostics are therefore more accessible, accurate, less expensive, and present less risk to the clinician than current methodologies. 

TECHNOLOGIES FOR SALIVA-BASED DIAGNOSTICS 

In 2002, the NIDCR initiated a concerted research effort in the area of salivary diagnostics and progress is currently moving toward technologically viable systems, which will lead to commercialization. NIDCR funded seven U01 (a designation for a large National Institutes of Health research grant) to develop microfluidics and microelectricomechanical systems (MEMS) for salivary diagnostics. MEMS are integrating systems consisting of mechanical elements, sensors, actuators, and electronics on a common silicon substrate (a substance upon which an enzyme acts) developed through microfabrication technology. These systems use a small sample and a reagent (substance used to produce a chemical reaction so as to detect, measure, or produce other substances) volumes coupled with integrated detection methods to perform analyses. The seven NIDCR-supported UO1 awards focused on the development of microfluidic and MEMS technologies for measuring DNA, gene transcripts (mRNA), proteins, electrolytes, and small molecules in saliva as well as overall profile correlates of a particular disease state, such as cardiovascular disease and cancer. 

DIAGNOSTIC MOLECULAR TARGETS IN SALIVA 

In 2003, NIDCR funded three UO1 awards aiming to comprehensively identify and catalog human salivary proteins from the three major salivary glands. The human salivary proteome is a resource in elucidating disease pathogenesis and evaluating the influence of medications on the structure, composition, and secretion of all salivary secretory constituents. 

The envisioned end product of the NIDCR program is a single human salivary proteome with input from all three groups. The first draft of the human salivary proteome should be available in 2007. This first comprehensive list of all salivary secretory components will help create the periodic table of the parotid, submandibular, and sublingual secretory components. 

UCLA COLLABORATIVE ORAL FLUID DIAGNOSTIC RESEARCH CENTER 

The UCLA School of Dentistry is engaged in both the technology development and the salivary proteome initiatives for salivary diagnostics. During the past 3 years, we have established the UCLA Collaborative Oral Fluid Diagnostic Research Center to develop the platform of using nano/micro technology to detect salivary protein and genomic biomarkers for point of care applications of high impact human diseases. 

 
Figure 4
UCLA's Oral Fluid NanoSensor Test (OFNASET).
 

For salivary diagnostic technology development, we partnered with engineers at the UCLA School of Engineering who are pioneers in the development of micro- and nano-electrical mechanical systems (MEMS and NEMS) biosensors that exhibit amazing sensitivity and specificity for analyte detection, down to single molecule level.4,5 Our research consortium has established a committed collaboration toward the development of MEMS/NEMS biosensors for the real time, ultrasensitive, and ultraspecific detection of salivary diagnostic analytes. Our prediction is that in less than 2 years, a lab-on-a-chip prototype will be available for research as well as patient applications.6 The envisioned product is the Oral Fluidic NanoSensor Test (OFNASET). The OFNASET is a handheld, automated, easy-to-use integrated system that will enable simultaneous and rapid detection of multiple salivary protein and nucleic acid targets (Figure 4). This saliva biomarker detector can be used in a dental or health care provider’s office for point-of-care disease screening and detection. In addition to the UCLA technologies, a number of other active groups are developing microfluidic technology platforms aiming for point of care multiplex detection of salivary analytes (visit http://nidcr.bioeng.washington.edu/Public/Funded_Projects2.htm for more information). Each of these engineering groups is spearheading technologies that will lead to the eventual commercialization of saliva-based point of care devices for detection of clinical diseases. 

To fully use the diagnostic potential of saliva, the informative components must be deciphered and cataloged. Comparison of such a catalogue with a disease population reveals diagnostic signatures that can discriminate between normal and diseased individuals. The salivary proteome presents such a resource. The UCLA group has already identified 309 proteins in human saliva.7 We have begun to make translational discoveries into the salivary proteome for patients with oral cancer8 and Sjögren’s syndrome (Shen Hu, PhD, unpublished data, 2006). 

Our laboratory has recently made the discovery that discriminatory and diagnostic human mRNAs are present in saliva of normal and diseased individuals. The salivary transcriptome (the collection of mRNAs) presents an additional valuable resource for disease diagnostics. Our first report of the salivary transcriptome, demonstrating that the normal salivary transcriptome consists of less than 3,000 mRNAs.9 Of particular value is that of the 3,000 mRNAs, 180 are common between different normal subjects, constituting the normal salivary transcriptome core (NSTC). To demonstrate the diagnostic and translation potential of the salivary transcriptome, saliva from head and neck cancer patients were profiled and analyzed. Four genes from the NSTC (IL8, OAZ1, SAT, and IL1B) were able to discriminate and predict if a saliva sample was from a cancer or normal subject with a sensitivity and specificity of 91% respectively. While head and neck cancer was used as the first proof-of-principle disease for salivary transcriptome diagnostics, data will soon be available for systemic diseases. These data, while early and exploratory, provide sufficient rationale and demonstrate the urgent need to fully explore salivary transcriptome diagnostic for major human disease translational applications. Adding to this urgency is our recent finding that the serum transcriptome from the same patients we examined for their salivary transcriptome yield 4 RNA biomarkers that have a sensitivity and specificity of 91% and 71% respectively (ROC= 0.88), demonstrating clearly that for oral cancer detection, saliva transcriptome diagnostics has a slight edge over blood.10 

FUTURE PERSPECTIVES 

While it is clear that there is a national agenda to turn salivary diagnostics into a clinical reality, much work needs to be done before this vision can be realized. Definitive disease-associated salivary biomarkers (proteins and genetic) need to be identified that can be used in conjunction with the technology platforms for salivary diagnostics. The scientific community is poised to develop and validate saliva-based tests as a chairside, portable, and multiplexible devices for diagnostic applications. Collectively, technology platform advancements and the identification and validation of discriminatory suites of salivary biomarkers for disease diagnostics represent the necessary marriage to propel saliva diagnostics into a clinical reality. 


 

David T. Wong, DMD, DMSc, is professor and associate dean of Research at the University of California, Los Angeles (UCLA) School of Dentistry. He is also director of the Dental Research Institute, professor in the Henry Samueli School of Engineering, and professor in the Division of Head and Neck Surgery/Otolaryngology both at UCLA. He has authored 129 peer reviewed scientific publications. Wong’s research is well-funded by the National Institutes of Health including two collaborative U01 grants on salivary technology development and salivary proteome. He is one of the country’s leading scientists in oral/head and neck cancer as well as salivary diagnostic research. 

Acknowledgement 

Supported by PHS grants UO1 DE-15018, UO1 DE-16275, RO1 DE-15970, and the UCLA Jonsson Comprehensive Cancer Center.

References
 

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6. St John MA, Li Y, Zhou X, et al. Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg . 2004;130:929-935.
7. Hu S ,Xie Y ,Ramachandran P , et al. Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry. Proteomics . 2005;5:1714-1728.
8. Hu S, Yu T, Xie Y, et al. Salivary proteomic biomarkers for human oral cancer. Cancer Genomics and Proteomics . In press.
9. Li Y, Zhou X, St John MA, Wong DT. RNA profiling of cell-free saliva using microarray technology. J Dent Res . 2004;83:199-203.
10. Li Y, Elashoff D, Oh M, et al. Serum circulating human mRNA profiling and its utility for oral cancer detection. J Clin Oncol . 2006;24:1754-1760.
 


From Dimensions of Dental Hygiene. July 2006;4(7): 14-17.

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