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    Saliva as a diagnostic tool

    July 22, 2015
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    Salivary diagnostics holds tremendous potential for the diagnosis, prognosis, and monitoring of cancer.1 The specimen can be obtained simply, inexpensively, and less invasively than blood and thus can be collected without special training.1-3 Saliva is a complex mixture including components such as salivary gland secretions, mucosal transudations, desquamated epithelial cells, expectorated bronchial and nasal secretions, bacteria and bacterial products, viruses, and food debris.1 Due to this complexity, saliva contains a rich variety of potential biomarkers including proteins, enzymes, antibodies, DNA, RNA, and cytokines.2,4

    In general, there are three methods for collecting saliva: salivary rinses, whole saliva, and stimulated saliva.2,3 A potential advantage of oral rinse collection methods is that the specimen contains whole saliva; it also has had contact with multiple sites including the oropharynx, oral cavity, and parts of the larynx and hypopharynx, whereas whole saliva or stimulated saliva only permit contact with the anterior oral cavity. Furthermore, concentrations of whole saliva components can vary greatly with hydration level. Since there is more contact with surfaces that contain secreted proteins, it is quite possible that the relative protein composition of the oral rinse would be different and perhaps more favorable than that of whole or stimulated saliva.2

    Salivary diagnostics for cancer

    Whole saliva contains serum components and therefore may be used for diagnosis, monitoring, and prognostic determination of cancers outside the mouth and throat, as recently reviewed by Malathi et al.1 In their review, they found that cerbB-2 and CA15-3 were differentially expressed in the saliva of women with breast cancer compared to healthy controls. Long noncoding RNA (lncRNA) are associated with lung, breast, and prostate carcinomas. Salivary mRNA markers have been defined for both lung and ovarian cancer. CA 125 is a tumor-associated antigen that is differentially expressed in serum and saliva of patients with oral, breast, and ovarian tumors, and salivary PSA levels correlate with serum PSA levels in patients with prostate cancer and therefore may become a useful marker of this disease.1

    Oral cancer

    In a study involving patients in India, oral cancer screening reduced oral cancer mortality by more than 80 percent in tobacco and/or alcohol users.5 However, this method of screening, visual inspection followed by tissue biopsy, has only 64 percent sensitivity for oral cancer6 and 31 percent specificity for oral dysplasia or cancer.7 Many molecular tests, hypermethylation, RNA, and protein-based panels, are under development, but not validated.8-11 Thus there is a need for something better, and saliva is becoming the biologic sample of choice for head and neck cancer screening.

    Work on salivary biomarkers for oral cancer detection has been extensively reviewed by Cheng et al.11 Markers include non-organic compounds; proteins including CD44, IL-8, cyclin D, and many others; DNAs including p53; mRNA including Il-8, OAZ1, and SAT, among others; microRNAs; oxidative stress-related molecules including peroxidase and superoxide dismutase; metabolomics; glycosylation-related molecules; and enzymes such as telemorase.11 Other technologies that use dyes, autofluorescence, or exfoliative cytology as adjuncts to the physical exam are used in clinical practice, but have not improved early detection rates.12,13

    Risk assessment in clinical practice

    Of the many oral cancer biomarkers studied, only a few are being translated to clinical practice. Among them are CD44, a cell surface transmembrane glycoprotein involved in cell proliferation and migration.14,15 CD44 is also a key tumor initiation marker16 that is over-expressed in the earliest stages of carcinogenesis.17,18 Soluble CD44 (solCD44), released by proteinases, is detectable in body fluids.19,20 Total protein, together with CD44, has also been shown to be an effective tumor marker. Both can be measured with simple, inexpensive assays.21-24

    Prior work shows that the combination of solCD44 and protein levels in oral rinses can distinguish head and neck squamous cell carcinoma (HNSCC) cases from controls.22-23 More recent work suggests that sensitivity can reach 88 percent for stage I-III cancer and specificity as high as 95 percent depending on the population studied (unpublished data). Recently, this technology has been converted to a lateral flow test strip point-of-care and a laboratory test, which will be commercially available soon. The inclusion of a cancer stem cell marker allows assessment of risk sufficiently early that reversal of carcinogenesis via behavioral change could be possible even before a lesion is clinically identified.

    Separate from CD44 and total protein, another group has performed a validation of the mRNA markers at the University of Michigan, Michigan State University, and the St. Johns Providence Health System in Detroit using technology developed by Dr. Wong at UCLA for the detection of oral cancer. The validation of these biomarkers demonstrated their feasibility in the discrimination of oral squamous cell carcinoma (OSCC) from healthy controls. In the five cohorts studied, the increase in IL-8 and SAT were statistically significant and remained top performers in terms of sensitivity and specificity. However, individual cutoff values for each of these markers and for the combined predictive model need to be further defined in large clinical studies.10 This work has been cited in the development of a risk stratification test recommended for use by the clinician only after suspicious visible lesions are observed and additional testing is warranted. The exact biomarkers used in the risk stratification system are not yet disclosed, to our knowledge.

    One saliva-based risk assessment tool for HNSCC currently available is for oral human papillomavirus (HPV) DNA testing. HPV infection is rapidly becoming the most important risk factor for cancers of the oropharynx, with the incidence of HPV+ oropharyngeal cancer rising in the United States.25-26 This HPV is a salivary test that assesses an individual’s risk of developing HPV-related oral cancers by identifying various HPV oncogenic genotypes. However, it is uncertain as to whether the HPV detected by this test shows whether the infection is past or current or even one that will eventually develop into HNSCC, whereas studies show usage of CD44 and total protein able to detect risk of the onset of HNSCC regardless of etiology. As a result, the usefulness of an assay detecting only HPV infection in assessing risk has yet to be determined.27

    References

    1. Malathi N, Mythili S, Vasanthi HR. Salivary diagnostics: a brief review. ISRN Dentistry. Published online 2014 Jan 29. doi:  10.1155/2014/158786 Accessed May 24, 2005.
    2. Franzmann EJ, Reategui EP, Pereira LH, et al. Salivary protein and solCD44 levels as a potential screening tool for early detection of head and neck squamous cell carcinoma. Head Neck. 2012;34(5): 687-695.
    3. Arellano M, Jiang J, Zhou X, et al. Current advances in identification of cancer biomarkers in saliva. Front Biosci (Schol Ed). 2009;1:296-303.
    4. T. Pfaffe, J. Cooper-White, P. Beyerlein, K. Kostner, C. Punyadeera. Diagnostic potential of saliva: current state and future applications. Clin Chem. 2011;57(5):675–687.
    5. Sankaranarayanan R, Ramadas K, Thara S, et al. Long term effect of visual screening on oral cancer incidence and mortality in a randomized trial in Kerala, India. Oral Oncol. 2013;49:314-321.
    6. Brocklehurst P, Kujan O, Glenny AM, et al. Screening programmes for the early detection and prevention of oral cancer. Cochrane database of systematic reviews. 2010:CD004150.
    7. Epstein JB, Güneri P, Boyacioglu H, Abt E. The limitations of the clinical oral examination in detecting dysplastic oral lesions and oral squamous cell carcinoma. J Am Dent Assoc. 2012;143:1332-1342.
    8. Hu S, Arellano M, Boontheung P, et al. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res. 2008;14(19);6246-6252.
    9. Carvalho AL, Jeronimo C, Kim MM, et al. Evaluation of promoter hypermethylation detection in body fluids as a screening/diagnosis tool for head and neck squamous cell carcinoma. Clin Cancer Res. 2008;14:97-107.
    10. Elashoff D, Zhou H, Reiss J, et al. Prevalidation of salivary biomarkers for oral cancer detection. Cancer Epidemiol Biomarkers Prev. 2012;21:664-672.
    11. Cheng YL, Rees T, Wright J. A review of research on salivary biomarkers for oral cancer detection. Clin Transl Med. 2014;3:3.
    12. Lingen MW, Kalmar JR, Karrison T, et al. Critical evaluation of diagnostic aids for the detection of oral cancer. Oral Oncol. 2008;44:10-22.
    13. Balevi B. Assessing the usefulness of three adjunctive diagnostic devices for oral cancer screening: a21. Screaton GR, Bell MV, Jackson DG, et al. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA. 1992;89:12160-12164.
    14. Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules to signaling regulators. Nature Rev Mol Cell Biol. 2003;4:33-45.
    15. Perez A, Neskey DM, Wen J, et al. CD44 interacts with EGFR and promotes head and neck squamous cell carcinoma initiation and progression. Oral Oncol. 2013;59(4):306-313.
    16. Prince ME, Sivanandan R, Kacsorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA. 2007;104:973-978.
    17. Hirvikoski P, Tammi R, Kumpulainen E, et al. Irregular expression of hyaluronan and its CD44 receptor is associated with metastatic phenotype in laryngeal squamous cell carcinoma. Virchows Arch. 1999;434(1):37-44.
    18. Ioachim E, Assimakopoulos D, Goussia AC, et al. Glycoprotein CD44 expression in benign, premalignant and malignant epithelial lesions of the larynx: an immunohistochemical study including correlation with Rb, p53, Ki-67 and PCNA. Histol Histopathol. 1999;14:1113-1118.
    19. Dasari S, Rajendra W, Valluru L. Evaluation of soluble CD44 protein marker to distinguish the premalignant and malignant carcinoma cases in cervical cancer patients. Med Oncol. 2014 2014;31(9):139.
    20. Kajita M, Itoh Y, Chiba T, et al. Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J. Cell Biol. 2001;153(5):893-904.
    21. Franzmann EJ, Reategui EP, Carraway KL, et al. Salivary soluble CD44: a potential molecular marker for head and neck cancer. Cancer Epidemiol Biomarkers Prev March 2005;14;735.
    22. Franzmann EJ, Reategui EP, Pereira LH, et al. Salivary protein and solCD44 levels as a potential screening tool for early detection of head and neck squamous cell carcinoma. Head Neck. 2012;34(5): 687–695.
    23. Pereira LH, Adebisi IN, Perez A, et al. Salivary markers and risk factor data: a multivariate modeling approach for head and neck squamous cell carcinoma detection. Cancer Biomark. 2011;10(5):241-249.
    24. Franzmann EJ, Reategui EP, Pedroso F, et al. Soluble CD44 is a potential marker for the early detection of head and neck cancer. Cancer Epidemiol Biomarkers Prev 2007;16(7):1348-1355.
    25. Gillison ML, Broutian T, Pickard RK, et al. Prevalence of oral HPV infection in the United States, 2009-2010. JAMA. 2012;307(7):693-703.
    26. D’Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med. 2007;356:1944-1956.
    27. Arunkumar S, Krishna AJS, Burde KN and Shakunthala GK. Developments in diagnostic applications of saliva in oral and systemic diseases—a comprehensive review. J Scientific and Innovative Research 2014;3(3):372-3787.

    Disclosures: The University of Miami and Dr. Franzmann hold intellectual property used in the study and have the potential for financial benefit from its future commercialization. Dr. Franzmann is the Chief Scientific Officer, consultant and an equity holder in Vigilant Biosciences, licensee of the IP used in the study.