You must be signed in to read the rest of this article.
Registration on CDEWorld is free. Sign up today!
Forgot your password? Click Here!
Oral cancer continues to have a significant tragic effect on the health, wellness, and mortality of many Americans. The American Cancer Society has projected that there were more than 51,540 new cases in 2018 with at least 10,030 deaths.1 Despite a heightened awareness of health professionals, these numbers have continued to increase annually. The entire dental profession, including all team members, has an opportunity and a responsibility to offer guidance to patients to be oral-cancer-free.
An effective oral cancer screening, which may be performed by trained team members, requires only a few minutes. This brief examination can positively affect a patient's health and help him or her appreciate the care that can be provided in the dental setting. In addition, the dental team can educate patients about oral cancer risk factors and offer them available preventive interventions. It is important that all health professionals and payers for oral health services collaborate to create an equitable and meaningful strategy that will significantly reduce, or eliminate, oral cancer. This is especially imperative in light of the emerging value-based payment health system that continues to grow to include a broad spectrum of providers and payers.
Although significant efforts have been made to increase the rate of early diagnosis, the 5-year survival rates and prognoses of oral squamous cell carcinoma (OSCC) have not improved due to delayed diagnosis.2 Delayed diagnosis is a result of many factors, such as a lack of or inadequate screening, financial barriers to proper diagnostic tests and biopsies, lack of education and action on the part of healthcare providers, and lack of action by patients due to absence of pain. Timely detection of OSCC and treatment at the earliest stages are essential for improved prognosis and survival rates.
Current methodologies used in diagnosis and detection of OSCC require biopsy to verify the existence of OSCC; however, photodynamics, vital staining, and cytology (Papanicolaou smear and brush biopsy) are increasingly being utilized to assist in early detection. Moving forward, use of salivary biomarkers and metabolomics analysis may aid in early detection, offering non-invasive, cost-effective methodologies to evaluate the physiological condition and provide information on pathology in its earliest stages.3
The brush biopsy is a relatively painless procedure that captures the deeper epithelial cells on the brush bristles, and the brush is sent to a pathology laboratory. This method helps diagnose white lesions in the mouth, overcoming some of the problems found in the use of pap smears. For example, pap smears were unable to find dysplastic cells in oral leukoplakias, because oral white lesions have a thicker keratin layer than their cervical counterparts.4 The brush biopsy, however, is not a true diagnostic tool and cannot provide a definitive diagnosis. An incisional biopsy is a more suitable method for obtaining a definitive diagnosis.5
Early detection of oral disease via a comprehensive examination of the oral cavity and surrounding structures is still one of the most effective means of timely management of systemic disease. Moreover, a complete examination that includes a thorough visual and tactile oral cancer screening test can be impactful. Because oral diseases and mucosal abnormalities may lead to malignancies, it is vital for oral health providers to be aware of the emerging roles that the human papillomavirus (HPV), fluorescent technology, and salivary biomarkers play in the prevalence and diagnosis of oral cancer and/or oropharyngeal cancers.
Human Papillomavirus and Head, Neck, and Oral Cancers
The HPV is the most common sexually transmitted disease in the United States.6 The main mode of transmission of HPV is skin to skin, and, therefore, in addition to vaginal, anal, and oral sex, the virus can be transmitted through open-mouth kissing and sharing inanimate objects like eating utensils and cups. While the Centers for Disease Control and Prevention (CDC) reports that 80% of Americans will be exposed to the HPV during their lifetime, approximately 99% of these individuals will clear the infection because of a healthy immune system.6 However, those who do not clear the infection are at a very high risk for genital warts and even cancer. In addition to cervical cancer, HPV also can cause cancer of the vagina, penis, anus, and mouth and throat (also known as oropharyngeal). While strains HPV-16 and HPV-18 have been strongly linked to cervical cancer, there are approximately 40 strains of HPV that affect the mouth and throat, causing cancer.1
There is no way to identify which people who have HPV will develop cancer (Figure 1). It is estimated that HPV is currently associated with more than 9,000 cases of head and neck cancers each year in the United States. The disease is four times more common in men than in women. Even though the association between HPV and oropharyngeal cancer was first proposed in 1983,7 a recent meta-analysis demonstrates a causal relationship between HPV DNA and oral cancer.8 It is possible that tonsillar crypts and deep periodontal pockets may serve as reservoirs, hence the increased prevalence in tonsillar cancers related to HPV. At present, there are no tests available to determine one's HPV status, nor are there any approved tests to identify HPV in the mouth or throat. However, there is an HPV vaccine that is safe and effective; it protects against diseases, including cancers, that are caused by the HPV when given in the recommended age groups.
The CDC recommends that 11- to 12-year-old boys and girls get two doses of HPV vaccine to prevent cervical and other less common genital cancers. There are currently three vaccines approved by the US Food and Drug Administration to protect against HPV, one of which is available in the United States. Additionally,in October 2018 the FDA approved a supplemental application for Gardasil 9 (HPV 9-valent vaccine, recombinant) expanding the approved use of the vaccine to include women and men aged 27 through 45 years. The vaccines protect against HPV types 16 and 18, which have been shown to be linked to cervical, vulvar, or anal cancer. Two of the vaccines also protect against strains 6 and 11, which are known to cause genital warts. It is possible that the HPV vaccine might also prevent head and neck cancers, as the vaccine prevents the initial infection with HPV types that can cause head and neck cancers.6 Multiple large studies completed in 2013, 2014, and 2016 have shown that these vaccines are as safe as any other vaccine and do not impact fertility.9
While any and all vaccines have side effects, the side effects that are possible with the HPV vaccine are so rare that, to date, there have been no reports of serious side effects. The CDC reports that HPV causes 70% of oropharyngeal cancers in the United States. Of these, 60% are caused by HPV-16, which is preventable with the HPV vaccine. Now more than ever, it is important for general dental providers to join forces with pediatric physicians to discuss the HPV vaccine and make appropriate recommendations.
Because HPV-associated oropharyngeal cancers have recently been increasing somewhat dramatically, healthcare providers, both medical and dental, should be knowledgeable about sexually transmitted diseases and high-risk sexual behaviors and comfortable discussing possible preventive strategies and solutions with their patients. Healthcare providers should look to existing frameworks that literature has proven successful as references to deliver an effective and meaningful conversation with parents and patients about HPV-related carcinomas.10,11
Visualization of Oral Tissue Fluorescence
Despite the oral cavity being a readily accessible region for direct visualization and cancer risk factors being well known, a significant amount of OSCCs still are diagnosed late in stage III or stage IV. Delayed diagnosis contributes to the 5-year survival rate being 57% and the worsening morbidity and mortality related to the disease.12 (According to Surveillance, Epidemiology, and End Results [SEER]program data, this rate is around 65%.13) Conventional examination of the oral cavity with incandescent white light is still the gold standard for OSCC screening and follow-up. This examination includes assessing for changes in surface texture, color, size, mucosal integrity, and mobility.14
The oral mucosa consists primarily of two layers: epithelium and stroma. The epithelium is stratified squamous tissue that comprises basal intermediate and superficial squamous cells. The stroma separates the epithelium from the basement membrane and consists of connective tissue, mostly collagen. The stroma also contains capillaries, and a surface layer of keratin may be present in varying layers. Certain types of oral mucosa are naturally keratinized, while other sites can become keratinized due to chronic irritation or because of other disease processes.
Each cell in a human contains molecules capable of self-fluorescence, especially when they are activated by specific light waves.15 The amount of fluorescence given off from living tissue is very small. However, if violet or blue light is used in a darkened room, and the clinician wears glasses that filter out virtually all reflected light and allows only transmission of the wavelength(s) of fluorescing tissues, the autofluorescence can be easily seen. The wavelengths (excitation emission matrix [EEM]) that excite the greatest fluorescence in oral mucosa range from 400 nm to 460 nm, ie, violet and blue light.
Reducing mortality and morbidity for OSCC is dependent mainly on the recognition of "oral potentially malignant disorders," because about 50% of OSCCs are preceded by these disorders.14 The association between cancer development and the loss of normal tissue fluorescence has been reported for many tissues and organs.16,17 Essentially, loss of autofluorescence is believed to reflect the complex and morphological and biochemical changes typical of squamous epithelial carcinogenesis.18,19
Bio- or autofluorescent technology (Figure 2) is a supportive mechanism using natural tissue fluorescence to enhance the visualization of the oral mucosa, or the region of inspection, to help enable the earliest possible discovery of potentially malignant lesions.16,20 Autofluorescent technology uses the fluorescence from fluorophores naturally present in the tissue to identify abnormalities. Fluorophores include various amino acid complexes, collagen, keratin, and coenzymes flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH).18 Being able to visualize early dysplastic changes in soft tissues in the oral cavity can allow clinicians to treat abnormal tissue variations before irreversible changes develop. This means completely compromising the entire basement membrane. Therefore, this method may add sensitivity to the oral examination and be an effective adjunct to high-risk patients.
Proteins in the tissue absorb autofluorescent light, enabling a visual reaction by emission of another wavelength. The wavelength emitted (405 nm to 460 nm) may have some diagnostic significance.18 The role of direct fluorescent visualization is four-fold: (1) discovery (screening); (2) biopsy guidance; (3) surgical margin delineation; and (4) surveillance and monitoring. Autofluorescent technology, also known as narrow band illumination, complements rather than replaces what can be seen with the naked eye and felt tactilely. It is fundamentally different from white light reflectance.18 To effectively use fluorescent technologies one must understand normal tissue variations as well as inflammatory tissue dynamics, traumatic tissue, and some underlying diseases that can mimic dysplastic tissue variations.17,21 Briefly, loss of autofluorescence is believed to reflect the complex and morphological and biochemical changes that are typical of squamous epithelial carcinogenesis.17-19,21
Diffuse reflectance is how the naked eye perceives objects under white light. The photons of light actually enter an object, get scattered or bounced around inside the object, and then come back out of a person's eye. These waves do not get absorbed first. White light is a mixture of all wavelengths of the visible light spectrum (ie, light blue, green, yellow, and red). Short wavelengths like blue light are strongly absorbed by the mucosal tissue.21 Almost all of the blue photons are fully absorbed, and very few make it back outside the tissue. When tissue is illuminated with light of an appropriate wavelength, also known as the EEM (or intensity of the light-emitting diode [LED] light), the light enters the tissue just as it does for reflectance, but gets absorbed by special naturally occurring molecules in the tissue called fluorophores. These fluorophores absorb the blue excitation light and then re-emit light at a longer wavelength. Blue light excites fluorophores in both the epithelium and the stroma. Natural fluorescence from tissue is relatively dim, much less bright than the blue reflected light. Natural fluorescence can be seen by the blocking of the brighter blue light utilizing filtering systems.21 These filtering systems optimize the contrast between normal and abnormal tissues. Fluorophores in the epithelium and stroma fluoresce green when illuminated with blue light. Tooth enamel will fluoresce more strongly than any other structure in the oral cavity.
Why is fluorescence a useful tool in helping to screen oral soft tissue? Certain biochemical, morphological, and environmental changes can accompany the disease process. The natural absorptive properties of the soft tissue can be altered with a net result of a change in fluorescence. Autofluorescence is always evaluated in relation to adjacent tissue in the field of view.15,22,23
One of the main fluorophores that is excited by blue light in the oral mucosa is FAD, which is thought to be the major contributor to epithelial fluorescence under blue light excitation. It is a coenzyme involved in the Krebs cycle and is correlated with metabolic activity in cells. When a cell is actively metabolizing there is a lower concentration of FAD. Therefore, highly dysplastic tissue behavior will correlate with increased metabolic activity, lower concentrations of FAD, and a decrease in dark violet or black fluorescence levels (longer wavelength being emitted).24,25 Thus, metabolism in the cellular map is a key component in fluorescent light behavior. Both FAD and NADH are correlated with metabolic activity in cells. Only FAD is excited by blue light. The increase in glucose oxidation will correlate with an increase in NADH and a decrease in FAD, as previously mentioned.16,26
Another main fluorophore to stromal fluorescence is collagen along with the collagen crosslinks that help maintain the structural integrity of the collagen matrix. Collagen crosslinks fluoresce when excited by blue light. As dysplasia of tissue progresses the collagen matrix starts to break down and can allow invasion of the basement membrane; thus, a decrease in fluorescence activity occurs.16,26 An increase in blood supply in the site of heightened cellular activity of the dysplastic cells will result in additional micro-vascularization in the stroma and, thus, increase the absorption of light. This, in turn, results in a net decrease in fluorescence as seen by the naked eye.27,28
The implementation and utilization of bio-fluorescent technologies requires a learning curve. Training in recognition of normal versus abnormal tissue under fluorescence is vital. Normal fluorescent patterns must not be misconstrued as dysplastic. The hard palate will have a more homogeneous color, usually pale green, than the tongue because it contains so many tiny papillae. Filliforms will fluoresce more brightly than fungiforms, because they have much less fluorescence due to the infusion of blood. Nonetheless, fluorescent technology has many benefits, as it is a non-invasive technique that requires no staining or rinse, it does not interfere with or compromise any other diagnostic tests, treatment, or protocols, and is quick and easy to use with proper training. Finally, it can be a cost-effective way of collecting additional information toward a proper and early diagnosis.16
Biomarkers are molecular signatures that are unique to a certain disease (eg, OSCC). They have been defined by the World Health Organization as any substance, structure, or process that can be measured in the body or its products, and influences or predicts the incidence or outcome of a disease.28 A biomarker is an objectively measured and evaluated indicator of normal biologic processes, pathologic processes, or pharmacologic responses to therapeutic intervention.29
For instance, biomarkers such as interleukin-1b (IL-1b) and interleukin-8 (IL-8) are associated with detection of OSCC.30 Salivary biomarker analysis for OSCC is a major advancement in diagnosis and is rapidly growing the field of scientific research. This has led to the emergence of salivary biomarkers as a promising diagnostic and prognostic tool in OSCC (Figure 3).
However appealing, the idea that more biomarkers will increase screening and diagnostic accuracy is untrue.31 Fundamentally, biomarkers are based on the simple idea that diseases disrupt normal metabolism to produce chemical indicators unique to particular diseases.32 For instance, erythrocytic sedimentation rate (ESR) is a universal indicator of inflammation as well as certain types of cancers. But ESR cannot identify a specific disease.33 Gingival inflammation and oral cancer both elevate ESR, but which disease is responsible for high ESR is indeterminate without further testing.34,35 ESR, therefore, is not very specific. By indicating only that there is a problem, ESR thus exhibits many false positives for cancer and is inefficient as a screen for any particular disease. However, ESR can serve as a first pass to rule out a problem. In other words, a normal ESR does not qualify for the next round of screening, thereby allowing the screener to focus on high-risk patients and provide patients peace of mind. Patients with an elevated ESR (a positive test) will require additional testing to maximize accurate detection of specific diseases.
In a sequence of analytic steps, the information value of biomarkers for screening diseases may be maximized without requiring many biomarkers, leading to an endless search for perfect ones and associated preservation and handling costs. It is time to think in terms of screening efficiencies rather than large and expensive screening programs. The promise of personalized medicine could be made more available, accessible, and cost-effective to more patients while improving accuracy.
While clinicians may be faced with false negatives, these could be balanced by improved specificity through factoring negative likelihood ratios into detection engines. Each screening project should be concerned with the balance of false positives and false negatives based on the consequences to both individuals and the public of making those errors.
Future research should compare accuracy by the diachronic method versus counts of independent biomarkers against gold standards of disease biopsy. In subsequent analyses, the costs saved by using and submitting fewer biomarkers for biopsy should be compared to usual operating procedures emphasizing the discovery of more biomarkers viv-a-vis referring more quickly to biopsy. Costs of care and risks of biopsies with false positives should be measured in utilities, cost-effectiveness, quality-adjusted life-years, length of stay, lives saved, and life-years saved. The perspective should be defined within either payer, patient, and/or quality of the complete healthcare system.36-38
Living cells contain many metabolites, derived from various metabolic activities. These metabolites are the final products of cellular biochemical processes, including gene transcription, mRNA translation, protein synthesis, and metabolic enzymatic reactions. The comprehensive identification and quantification of these metabolites is called metabolomics. A research study by Wang et al revealed five salivary metabolites (biomarkers) that had significant sensitivity in the diagnosis of early stages of stage I and stage II OSCC: propionylcholine, acetylphenylalanine, sphinganine, phytosphingosine, and s-sarboxymethyl-l-cystine.36-38
In the modern biological era the "omics" approach is regarded as a new biomarker sighting tool that focuses on a large set of molecules. Genomics (genome), transcriptomics (transcriptome), proteomics (proteome), and metabolomics (metabolome) are the various techniques that help enable an easy and systematic understanding of the highly complex biological system. The "omics" approach is quite different from the conventional technique for studying complex biological systems. In view of this, "omics" studies are gaining importance in studying both normal physiological processes and pathological conditions in various clinical applications. Apparently, these technologies perform better in higher dimensional biological studies to explore the detailed description of conditions from genome to metabolome.39 The "omics"-based approaches can help clinicians understand the complex processes of oral cancer progression from pre-cancer to cancer state. From the diagnostic point of view, evaluating the altered metabolic pathways associated with the disease could be helpful for decreasing its mortality and morbidity.
While metabolomics research into OSCC is in its early stages, several findings have been noted, including some physiological functions. In oral cancer research, the metabolomics approach has offered various novel insights into cancer metabolism; eg, it has identified various cancer-specific metabolic pathways. In the future, metabolomic analysis of oral specimens might provide a wide range of novel information, leading to more accurate diagnosis, safer and more effective treatment, and preventive strategies of oral and systemic disorders.
While the use of advanced technologies continues to be studied and analyzed, the future of oral cancer diagnosis is very promising. There is much that healthcare providers can do now to actively combat oral, head, and neck cancer so it may be detected, diagnosed, and treated earlier.
First, oral healthcare providers should receive a thorough and proper oral cancer screening once a year if there are no risk factors. If there are risk factors, multiple screenings throughout the year should be completed. This speaks to providers not only taking better care of themselves and looking after their own health, but it also underscores the importance of healthcare providers following the same advice they give to their patients.
Second, oral healthcare providers should learn how to perform a thorough visual and tactile oral cancer screening on their patients. These screenings should be done on every patient in the dental office or clinic at least once a year, if not at every visit for those who are at moderate or high risk.
Third, it is critical that oral healthcare providers stay up to date on the risk factors related to oral, head, and neck cancer, and develop the skills to support patients with cessation of drugs and habits that increase the risk for these types of cancer. By educating patients about the risk factors and lifestyle habits that are related to increased risk for cancer, providers are helping raise the oral health literacy in their community.
About the Authors
Leonard B. Goldstein, DDS, PhD
Assistant Vice President for Clinical Education Development, Arizona School of Dentistry & Oral Health, A.T. Still University, Mesa, Arizona; The ATSU Center for the Future of the Health Professions
Mai-Ly Duong, DMD, MPH, MAEd
Assistant Professor, Arizona School of Dentistry & Oral Health, A.T. Still University, Mesa, Arizona; Private Practice, Phoenix, Arizona
Robert Levine, DDS
Director of Laser Dentistry, Arizona School of Dentistry & Oral Health, A.T. StillUniversity, Mesa, Arizona; Founder, Global Laser Oral Health, LLC, Scottsdale, Arizona
Jack Dillenberg, DDS, MPH
Dean Emeritus, Arizona School of Dentistry & Oral Health, A.T. Still University, Mesa, Arizona; The ATSU Center for the Future of the Health Professions
Queries to the author regarding this course may be submitted to firstname.lastname@example.org.
1. Cancer Statistics Center. Oral cavity and pharynx. American Cancer Society website. https://cancerstatisticscenter.cancer.org/#!/cancer-site/Oral%20cavity%20and%20pharynx. Accessed March 4, 2019.
2. American Cancer Society. Cancer Facts & Figures 2018. Atlanta, GA: American Cancer Society; 2018. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2018/cancer-facts-and-figures-2018.pdf. Accessed March 12, 2019.
3. Boguspour N, Goldstein LB. Oral cancer diagnosis: from biopsy to metabolomics. EC Dental Science. 2018;17(6):800-803.
4. Rajput DV, Tupkari JV. Early detection of oral cancer: PAP and AgNOR staining in brush biopsies. J Oral Maxillofac Pathol. 2010;14(2):52-58.
5. Sciubba JJ. Improving detection of precancerous and cancerous lesions. Computer-assisted analysis of the oral brush biopsy. U.S. Collaborative Oral CDx Study Group. J Am Dent Assoc. 1999;130(10):1445-1457.
6. National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention. Genital HPV Infection - CDC Fact Sheet. Centers for Disease Control and Prevention website. July 2017. https://www.cdc.gov/std/hpv/HPV-FS-July-2017.pdf. Accessed March 4, 2019.
7. Khangura RK, Sengupta S, Sircar K, et al. HPV involvement in OSCC: correlation of PCR results with light microscopic features. J Oral Maxillofac Pathol. 2013;17(2):195-200.
8. Chaitanya NC, Allam NS, Gandhi Babu DB, et al. Systematic meta-analysis on association of human papilloma virus and oral cancer. J Cancer Res Ther. 2016;12(2):969-974.
9. Human Papillomavirus (HPV). Questions and Answers. Centers for Disease and Prevention website. August 23, 2018. https://www.cdc.gov/hpv/parents/questions-answers.html. Accessed March 12, 2019.
10. Khariwala SS, Moore MG, Malloy KM, et al. The "HPV Discussion": effective use of data to deliver recommendations to patients impacted by HPV. Otolaryngol Head Neck Surg. 2011;153(4);518-525.
11. Lee JS, Somerman MJ. The importance of oral health in comprehensive health care. JAMA. 2018;320(4):339-340.
12. Carvalho AL, Nishimoto IN, Califano JA, Kowalski LP. Trends in incidence and prognosis for head and neck cancers in the United States: a site-specific analysis of the SEER database. Int J Cancer. 2005;114
13. Surveillance, Epidemiology, and End Results Program. Cancer Stat Facts: Oral Cavity and Pharynx Cancer. National Cancer Institute website. https://seer.cancer.gov/statfacts/html/oralcav.html. Accessed March 12, 2019.
14. Reibel J. Prognosis of oral pre-malignant lesions: significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med. 2003;14(1):47-62.
15. Williams PM, Poh CF, Hovan AJ, et al. Evaluation of a suspicious oral mucosal lesion.J Can Dent Assoc. 2008;74(3):275-280.
16. Pavlova L, Williams M, El-Naggar A, et al. Understanding the biological basis of autofluorescence imaging for oral cancer detection: high-resolution fluorescence microscopy in viable tissue. Clin Cancer Res. 2008;14(8):2396-2404.
17. Falk GW. Autofluorescence endoscopy. Gastrointest Endosc Clin N Am. 2009;19(2):209-220.
18. Roblyer D, Kurachi C, Stepanek V, et al. Objective detection and delineation of oral neoplasia using autofluorescence imaging. Cancer Prev Res (Phila). 2009;2(5):423-431.
19. Cånjău S, Todea DCM, Sinescu C, et al. Fluorescence influence on screening decisions for oral malignant lesions. Rom J Morphol Embryol. 2018;59(1):203-209.
20. De Veld DC, Witjes MJ, Sterenborg HJ, Roodenburg JL. The status of in vivo autofluorescence spectroscopy and imaging for oral oncology. Oral Oncol. 2005;41(2):117-131.
21. Awan KH, Morgan PR, Wamakulasuriga S. Evaluation of an autofluorescence based imaging system (VELscope™) in the detection of oral potentially malignant disorders and benign keratoses. Oral Oncol. 2011;47(4):274-277.
22. Poh CF, Zhang L, Anderson DW, et al. Fluorescence visualization detection of field alterations in tumor margins of oral cancer patients. Clin Cancer Res. 2006;12(22):6716-6722.
23. Truelove EL, Dean D, Maltby S, et al. Narrow band (light) imaging of oral mucosa in routine dental patients. Part 1: assessment of value in detection of mucosal changes. Gen Dent. 2011;59(4):281-289.
24. Farah C. Technologies enable early and accurate oral cancer detection. Dent Today. April 2016. https://www.dentistrytoday.com/news/todays-dental-news/item/902-technologies-enable-early-and-accurate-oral-cancer-detection?highlight=WyJvcmFzY29wdGljIiwib3Jhc2NvcHRpYydzIl0=. Accessed March 4, 2019.
25. Schwarz RA, Richards-Kortum RR, Gillenwater AM. Fluorescence and reflectance spectroscopy for detection of oral dysplasia and cancer. In: Wong BF, Ilgner J, eds. Biomedical Optics in Otorhinolaryngology. New York, NY: Springer; 2016:431-449.
26. Prahl S. Optical absorption of hemoglobin. https://omlc.org/spectra/hemoglobin/. Accessed March 4, 2019.
27. Zheng W, Li D, Zeng Y, et al. Two-photon excited hemoglobin fluorescence. Biomed Opt Express. 2010;2(1):71-79.
28. Bano S, David MP, Indira AP. Salivary biomarkers for oral squamous cell carcinoma: an overview. IJSS Case Rep Rev. 2015;1(8):39-45.
29. Spielmann N, Wong DT. Saliva: diagnostics and therapeutic perspectives. Oral Dis. 2011;17(4):345-354.
30. Santosh AB, Jones T, Harvey J.A review of oral cancer biomarkers: understanding the past and learning from the present. J Cancer Res Ther. 2016;12(2):486-492.
31. Kraemer HC. Evaluating Medical Tests: Objective and Quantitative Guidelines. 1st ed. Newbury Park, CA: Sage Publications, Inc.; 1992.
32. Schafer CA, Schafer JJ, Yakob M, et al. Saliva diagnostics: utilizing oral fluids to determine health status. Monogr Oral Sci. 2014;24:88-98.
33. Fuentes L, Yakob M, Wong DT. Emerging horizons of salivary diagnostics for periodontal disease. Br Dent J. 2014;217(10):567-573.
34. Feller L, Altini M, Khammissa RA, et al. Oral mucosal immunity. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;116(5):576-583.
35. Feller L, Altini M, Lemmer J. Inflammation in the context of oral cancer. Oral Oncol. 2013;49(9):887-892.
36. Wang Q, Gao P, Wang X, Duan Y.The early diagnosis and monitoring of squamous cell carcinoma via saliva metabolomics. Sci Rep. 2014;4:6802. doi: 10.1038/srep06802.
37. Ayanian JZ, Markel H. Donabedian's lasting framework for health care quality. N Engl J Med. 2016;375(3):205-207.
38. Menke JM, Roy D. Better disease screening with fewer biomarkers. Mesa, AZ: A.T. Still Research Institute. In press.
39. Wei J, Xie G, Zhou Z, et al. Salivary metabolite signatures of oral cancer and leukoplakia. Int J Cancer. 2001;129(9):2207-2217.